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AMPLIFICATION
CELL BIOLOGY
CLONING
MICROARRAYS
NUCLEIC ACID ANALYSIS
PROTEIN FUNCTION & ANALYSIS
QUANTITATIVE PCR
SOFTWARE SOLUTIONS
Contents
2
Introductions:
Bringing About Change
Sean Sanders
The Power of Mutagenesis
Patricia Sardina
4
Bypassing a Kinase Activity with an ATP-Competitive Drug
Feroz R. Papa, Chao Zhang, Kevan Shokat, Peter Walter
Science 28 November 2003 302: 1533-1537
12
Human Catechol-O-Methyltransferase Haplotypes Modulate
Protein Expression by Altering mRNA Secondary Structure
A. G. Nackley, S.A. Shabalina, I. E. Tchivileva, K. Satterfield,
O. Korchynskyi, S. S. Makarov, W. Maixner, L. Diatchenko
Science 22 December 2006 314: 1930-1933
17
An mRNA Surveillance Mechanism That Eliminates Transcripts
Lacking Termination Codons
Pamela A. Frischmeyer, Ambro van Hoof, Kathryn O’Donnell,
Anthony L. Guerrerio, Roy Parker, Harry C. Dietz
Science 22 March 2002 295: 2258–2261
24
Direct Demonstration of an Adaptive Constraint
Stephen P. Miller, Mark Lunzer, Antony M. Dean
Science 20 October 2006 314: 458–461
30
Technical Notes: Large Insertions: Two Simple Steps Using
Quikchange® II Site-Directed Mutagenesis Kits
About the Cover:
γ-Exonuclease binds to an end of double-stranded
DNA and degrades one of the strands in a
highly processive manner. The structure of the
exonuclease consists of a toroidal trimer (~94
angstroms in diameter) and is presumed to enclose
its substrate in the manner illustrated. [Image from
the cover of Science 277 (19 September 1997)]
Design and Layout: Amy Hardcastle
Copy Editor: Robert Buck
© 2007 by The American Association for the Advancement of Science.
All rights reserved.
1
Introductions
Bringing About Change
This new method of mutagenesis has considerable potential in genetic studies.… [S]pecific
mutation within protein structural genes will allow the production of proteins with specific amino
acid changes. This will allow precise studies of structure-function relationships, for example
the role of specific amino acids in enzyme active sites, and the more convenient production of
proteins whose action is species specific, for example the conversion of a cloned gene coding for
a lower vertebrate hormone to that for a human.
— closing paragraph of the seminal paper from Michael Smith’s lab first describing
site-directed mutagenesis1
The art of life lies in a constant readjustment to our surroundings.
— Okakura Kakuzo, Japanese scholar
I
t is a truism that, to survive, we must be able to change. This, too, is the essence of
evolution on the most fundamental biological level: without the innate ability for
DNA to undergo mutation, it would not be possible for organisms to adapt and survive
changes in their environment. Since humankind achieved an understanding of this mutagenesis process and its power, we have been trying to control it and bend it to our will.
First described in 1978 by Michael Smith at the University of British Columbia in
Vancouver,1 site-directed mutagenesis (SDM) has become an invaluable tool for molecular biologists. Originally named oligonucleotide-directed mutagenesis, the first experiments made use of short, 12-nucleotide strands of synthetic DNA containing a mismatch
to an intrinsic reporter gene of the bacteriophage ΦX174. Using these oligonucleotides,
together with DNA polymerase I from E. coli and T4 DNA ligase, Smith and his team
demonstrated that it was possible to introduce a permanent mutation in the circular DNA
of the phage, resulting in a phenotypic change. The prescient quote above shows the authors’ understanding of the manifold applications for the new methodology.
Since its development, a number of modifications and improvements have been made
to the SDM technique. The greatest advance came with the invention of the polymerase
chain reaction (PCR) in 1983. Since then, SDM and PCR have been inextricably linked,
a circumstance reflected in the 1993 Nobel in Chemistry being shared by Kary Mullis,
the pioneer of PCR, and Michael Smith. This advance made SDM both quicker and more
flexible and has enabled it to become a standard and necessary technique employed in
labs worldwide.
In this booklet, we bring together a collection of influential papers that all depended
on SDM for their success. As one of the most used techniques in molecular biology, it
plays an often unacknowledged–but essential–role in many research projects. There is little doubt that future refinements and improvements will be made to the technique, allowing for an even broader range of applications. As these modifications are commercialized
and made available to all scientists, new and innovative uses for them will be discovered,
facilitating enrichment of our knowledge in many areas of research, from basic cellular
processes to complex diseases such as neurodegenerative disorders and cancer.
Sean Sanders, Ph.D.
Commercial Editor, Science
1
C. A. Hutchison III, et al. Mutagenesis at a specific position in a DNA sequence. J. Biol. Chem. 253:6551-6560 (1978).
2
The Power of Mutagenesis
I
n vitro mutagenesis is a very powerful tool for studying protein structure-function
relationships, altering protein activity, and for modifying vector sequences to incorporate affinity tags and correct frame shift errors. A variety of mutagenesis techniques
have been developed over the past decade that allow researchers to generate point mutations, modify (insert, delete or replace) one or more codons, swap domains between
related gene sequences, and create diverse collections of mutant clones.
Mutagenesis strategies can be divided into two main types, random or site-directed.
With random mutagenesis, point mutations are introduced at random positions in a geneof-interest, typically through PCR employing an error-prone DNA polymerase (errorprone PCR). Randomized sequences are then cloned into a suitable expression vector,
and the resulting mutant libraries can be screened to identify mutants with altered or improved properties. By correlating changes in function to alterations in protein sequence,
researchers can create a preliminary map of protein functional domains.
Site-directed mutagenesis is the method of choice for altering a gene or vector sequence at a selected location. Point mutations, insertions, or deletions are introduced by
incorporating primers containing the desired modification(s) with a DNA polymerase in
an amplification reaction. In more complex protein engineering experiments, researchers
can design mutagenic primers to incorporate degenerate codons (site-saturation mutagenesis).
Stratagene, now an Agilent Technologies company, is a worldwide leader in developing innovative products and technologies for life science research. Our expertise in enzyme engineering has translated into the most efficient and easy-to-use mutagenesis kits
available today. Our QuikChange® Site-Directed Mutagenesis products provide the most
reliable and accurate method for performing targeted mutagenesis, saving time and valuable resources. These products have been cited in thousands of publications, including
the papers featured in this booklet. In addition, our GeneMorph® Random Mutagenesis
products offer an easy, robust method for creating diverse mutant collections.
Our mutagenesis product offering has been substantially enhanced by our newest
product introduction, the QuikChange® Lightning Site-Directed Mutagenesis Kit. This
kit offers significant time-savings by incorporating new, exclusive fast enzymes which
allow researchers to shorten the duration of the thermal cycling and digestion steps while
maintaining ultra-high fidelity and mutation efficiency.
The efficacy of mutagenesis as a tool for studying protein function and structure may
lead to new experimental therapeutic approaches to diseases such as cancer. Precise mapping of protein-protein interactions may ultimately lead to a greater understanding of
disease mechanics. This Science Mutagenesis Techniques Collection booklet illustrates
the utility of mutagenesis in a wide variety of research studies. We are pleased to have the
opportunity to sponsor this publication.
Patricia Sardina
Product Manager
Stratagene, An Agilent Technologies Company
3
Bypassing a Kinase Activity with an
ATP-Competitive Drug
Bypassing
a Kinase Activity with
Feroz R. Papa, * Chao Zhang, Kevan Shokat, Peter Walter
an ATP-Competitive Drug
1,3
2
2
3,4
Unfolded proteins in the endoplasmic reticulum cause trans-autophosphorylation
of1,3*the
bifunctional
transmembrane
kinase
Ire1, 3,4
which induces its endoFeroz R.
Papa,
Chao
Zhang,2 Kevan
Shokat,2 Peter
Walter
ribonuclease activity. The endoribonuclease initiates nonconventional splicing
of HAC1
messenger
RNA to trigger
the unfolded-protein
response (UPR). We
Unfolded
proteins
in the endoplasmic
reticulum
cause trans-autophosphorylation
of the
explored
the role of Ire1’s
kinase
domain
sensitizing
it through site-directed
bifunctional
transmembrane
kinase
Ire1,
which by
induces
its endoribonuclease
activity. The
mutagenesisinitiates
to the ATP-competitive
1NM-PP1.
Paradoxically,
rather
endoribonuclease
nonconventionalinhibitor
splicing of
HAC1 messenger
RNA to
trigger
than being
inhibited
by 1NM-PP1,
Ire1ofmutants
required
1NMthe unfolded
protein
response
(UPR). Wedrug-sensitized
explored the role
Ire1’s kinase
domain
by
PP1 as
a cofactor
for activation.
In the presence
of 1NM-PP1, drug-sensitized
sensitizing
it through
site-directed
mutagenesis
to the ATP-competitive
inhibitor 1NMIre1 bypassed
mutations
that inhibited
inactivateby
its1NM-PP1,
kinase activity
and induced
full
PP1. Paradoxically,
rather
than being
drug-sensitized
Ire1a mutants
UPR.
Thus, rather
than through
phosphorylation
per se,
a conformational
required
1NM-PP1
as a cofactor
for activation.
In the presence
of 1NM-PP1,
drugchange
the kinase
domain that
triggered
by occupancy
the active
withaafull
sensitized
Ire1in
bypassed
mutations
inactivate
its kinaseofactivity
and site
induced
ligand leads to activation of all known downstream functions.
UPR. Thus, rather than through phosphorylation per se, a conformational change in the
kinase domain triggered by occupancy of the active site with a ligand leads to activation
mRNA (u stands for uninduced), which enSecretory and transmembrane proteins traof all known downstream functions.
codes the Hac1 transcriptional activator necversing the endoplasmic reticulum (ER) duressary for activation of UPR targets (5).
ing their biogenesis fold to their native states
is constitutively
transcribed,
HAC1u mRNA
in thisecretory
compartment
This process isprofa- functional
domains
(4). The most
and (1).
transmembrane
N-terbut not translated, because it contains a noncilitated by a plethora of ER-resident activiteins
traversing
the
endoplasmic
minal
domain,
which
resides
in
the
ER
conventional translation-inhibitory intron
ties (the protein folding machinery) (2). Inreticulum
during
theircauses
biogensenses
elevated
levels
of unfolded
(6). Upon
activation,
Ire1’s
RNase
cleaves
sufficient
protein (ER)
folding
capacity
an lumen,
u
proteins.
ER excising
chaperesis
fold to their
native states
in this
commRNADissociation
at two specificofsites,
HAC1
accumulation
of unfolded
proteins
in the
ER ER
the intron
andbecome
3� exonsengaged
are relumen (a condition
referred is
to facilitated
as ER stress)
partment
(1). This process
from(7).
Ire1The
as 5�
they
by ones
joinedunfolded
by tRNA
ligaseis(8),
resulting
in
triggers of
a transcriptional
program called
proteins
thought
to
trigaand
plethora
ER-resident activities
(the with
spliced HAC1i mRNA (i stands for induced),
the UPR. In yeast, UPR targets include genes
protein
folding machinery) (2). Insuffi- ger
Ire1 activation (3).
which is actively translated to produce the
encoding chaperones, oxido-reductases,
cient
protein folding
capacity
causesERan Hac1
Ire1’s
most C-terminal
domain
is a regtranscriptional
activator,
in turn
upphospholipid
biosynthetic
enzymes,
accumulation
of unfolded
proteinsand
in prothe ulated
endoribonuclease
which
regulating
UPR target genes(RNase),
(5).
associated degradation
components,
ER
(a condition
referred
as ER hasAa functional
single known
substrate
yeast: the
kinase
domain in
precedes
teinslumen
functioning
downstream
in theto
secretory
RNaseudomain
cytosolic
of the ER
pathwayand
(3).triggers
Together,
UPR target activities
stress)
a transcriptional
pro- HAC1
mRNAon(uthestands
forside
uninduced),
membrane
(9). Activation
of Ire1
leads to its
afford called
proteins
through
ERtaran which
gram
thepassing
UPR. In
yeast,the
UPR
encodes
the Hac1
transcriptional
oligomerization in the ER membrane, folextended opportunity to fold and assemble
necessary for activation of
UPR
gets
include genes encoding chaperones, activator
lowed by trans-autophosphorylation
(9, 10).
properly, dispose of unsalvageable unfolded
u
targets
(5).
oxido-reductases,
phospholipid
biosynHAC1
mRNA
is
constitutively
Mutations of catalytically essential kinase
polypeptides, and increase the capacity for
thetic
enzymes, ER-associated degrada- transcribed,
but notortranslated,
because
it
active-site residues
mutations of
residues
ER export.
known toa nonconventional
become phosphorylated
prevent
yeast UPR and
is signaled
through
the ER
contains
translation-intionThe
components,
proteins
functioning
splicing
abrogate Ire1’s
UPR
HAC1u mRNA
stress sensorinIre1,
a single-spanning
ER hibitory
downstream
the secretory
pathway (3).
intron (6).
Uponand
activation,
signaling, demonstrating
that Ire1’s kinase
transmembrane protein with three functional
Together, UPR target activities afford pro- RNase
cleaves HAC1u mRNA
at two spephosphotransfer function is essential for
domains (4). The most N-terminal domain,
teins
thelumen,
ER ansenses
extended
excising
the11).
intron
5′
RNasesites,
activation
(4, 9,
Thus,(7).
Ire1The
comwhichpassing
resides through
in the ER
ele- cific
opportunity
to fold
and assemble
properly,
3′ exons
rejoined by tRNA
municates
an are
unfolded-protein
signalligase
from
vated levels of
unfolded
ER proteins.
Disso- and
i
the ER
to the cytosol
using HAC1
the lumenal
dociation ofofER
chaperones from
Ire1 aspolythey (8),
dispose
unsalvageable
unfolded
resulting
in spliced
mRNA
main
as
the
sensor
and
the
RNase
domain
as
become
engaged
with
unfolded
proteins
is
peptides, and increase the capacity for ER (i stands for induced), which is actively
the effector.toAlthough
important
for
thought to trigger Ire1 activation (3).
export.
translated
produce clearly
the Hac1
transcripthe circuitry of this machine, it is unknown
Ire1’s most C-terminal domain is a reguThe yeast UPR is signaled through the tional
activator, in turn upregulating UPR
why and how Ire1’s kinase is required for
lated endoribonuclease (RNase), which has a
u
ER
stress
sensor
Ire1,
a
single-spanning
target
genes
(5).RNase. To dissect the role of
activation of its
single known substrate in yeast: the HAC1
ER transmembrane protein with three Ire1’s
A functional
kinasewedomain
kinase function,
used aprecedes
recently
developed
us to sensitize
the
RNasestrategy
domainthat
onallows
the cytosolic
side
1
2
Department of Medicine, Department of Cellular
Ire1the
to specific
kinase inhibitors
(12).
of
ER
membrane
(9).
Activation
of
3
and Molecular Pharmacology, Department of Biobehavior of Ire1 mutants
chemistry and Biophysics, 4Howard Hughes Medical
Ire1Unexpected
leads to its
oligomerization in the
sensitized to an ATP-competitive drug.
Institute, University of California, San Francisco, CA
ER
membrane,
byattrans-auto745
94143–2200, USA.
The mutation
of Leufollowed
—situated
a conserved
phosphorylation
(9,
10).
Mutations
of
position
in
the
adenosine
5�-triphosphate
(ATP)–
*To whom correspondence should be addressed. Email: [email protected]
binding site—toessential
Ala or Glykinase
is predicted
to sencatalytically
active-site
S
4
sitize Ire1
butyl-3-nap
d]pyrimidin
an enlarged
wild-type k
fected by
partially o
Ire1, these
UPR sign
vivo repo
creased k
[Ire1(L745
relative to
Ire1(L745
approachin
(Fig. 1A).
Unexpe
to cells
Ire1(L745A
concentrat
1NM-PP1–
stead, 1NM
slightly (F
prise, 1NM
stored sig
Ire1(L745G
presents a
compound
an inhibito
tant enzym
UPR ac
by dithioth
cin (18); 1
indicating
directly af
over, the e
other stru
naphthylm
and 1-naph
ene group
the naphth
hibited wil
Activat
ATP-comp
tion of wh
sary in the
this end, w
mutation
D828A, w
due needed
as the Ire
was detect
wild-type c
detectable,
and could
Ire1(L745A
the UPR r
addition o
levels appr
tivation lev
binding of
Ire1 rende
www.sciencemag.org SCIENCE VOL 302 28 NOVEMBER 2003
residues or mutations of residues known
to become phosphorylated prevent HAC1u
mRNA splicing and abrogate UPR signaling, demonstrating that Ire1’s kinase
phosphotransfer function is essential for
RNase activation (4, 9, 11). Thus, Ire1
communicates an unfolded-protein signal
from the ER to the cytosol using the lumenal domain as the sensor and the RNase
domain as the effector. Although clearly
important for the circuitry of this machine,
it is unknown why and how Ire1’s kinase
is required for activation of its RNase. To
dissect the role of Ire1’s kinase function,
we used a recently developed strategy that
allows us to sensitize Ire1 to specific kinase inhibitors (12).
Unexpected behavior of Ire1 mutants sensitized to an ATP-competitive
drug. The mutation of Leu745—situated
at a conserved position in the adenosine
5′-triphosphate (ATP)–binding site—to
Ala or Gly is predicted to sensitize Ire1 to
the ATP-competitive drug 1-tert-butyl-3naphthalen-1-ylmethyl-1H-pyrazolo[3,4d]pyrimidin-4-ylemine (1NM-PP1) by
creating an enlarged active-site pocket not
found in any wild-type kinase (13). Most
kinases are not affected by these mutations, but others are partially or severely
impaired (14, 15). For Ire1, these substitutions markedly decreased UPR signaling (assayed through an in vivo reporter),
which is indicative of decreased kinase activity. Ire1(L745→A745) [Ire1(L745A) (16)]
showed a 40% decrease relative to the
wild-type kinase, whereas Ire1(L745G)
showed a >90% decrease, approaching
that of the Δire1 control (Fig. 1A)
Unexpectedly, the addition of 1NMPP1 to cells expressing partially active
Ire1(L745A) caused no inhibition, even
at concentrations that completely inhibit
other 1NM-PP1–sensitized kinases (15,
17). Instead, 1NM-PP1 increased reporter
activity slightly (Fig. 1A, bar 4). To our
further surprise, 1NM-PP1 provided significantly restored signaling to the severely crippled Ire1(L745G) (Fig. 1A, bar 6).
This result presents a paradox: An ATPcompetitive compound that was expected
to function as an inhibitor of the rationally
engineered mutant enzyme instead permits activation.
UPR activation strictly required induction by dithiothreitol (DTT) (Fig. 1) or
tunicamycin (18); 1NM-PP1 by itself had
no effect, indicating that it does not induce
the UPR by directly affecting ER protein
folding. Moreover, the effects of 1NMPP1 were specific: other structurally similar compounds, 2-naphthylmethyl PP1,
which is a regio-isomer, and 1-naphthyl
PP1, which lacks the methylene group between the heterocyclic ring and the naphthyl group, neither activated nor inhibited
wild-type or mutant Ire1 (18).
Activation rather than inhibition by
an ATP-competitive inhibitor raised the
question of whether the kinase activity is necessary in the 1NM-PP1–sensitized mutants. To this end, we combined
the L745A or L745G mutation with a
“kinase-dead” variant D828A, which
lacks an active-site Asp residue needed
to chelate Mg2+(11, 19). Whereas the
Ire1(L745A,D828A) double mutant was
detectable in cells at levels near those of
wild-type cells, Ire1(L745G,D828A) was
undetectable, probably because of instability, and could not be assayed. As expected,
Ire1(L745A,D828A) was unable to induce
the UPR reporter with DTT alone, but
the addition of 1NM-PP1 allowed activation to levels approaching 80% of the
wild-type activation level (Fig. 1B). This
suggests that the binding of 1NM-PP1 to
1NM-PP1–sensitized Ire1 renders the kinase activity dispensable.
Trans-autophosphorylation by Ire1 of
two activation-segment Ser residues (S840
and S841) is necessary for signaling (9),
prompting the question of whether this
requirement is also bypassed in 1NMPP1–sensitized mutants. As expected, the
unphosphorylatable Ire1 (S840A,S841A)
mutant was inactive in the absence and
presence of 1NM-PP1 (Fig. 1C). In
5
1 of two
and S841)
pting the
t is also
mutants.
ble Ire1
n the abFig. 1C).
ed Ire1
1(L745G,
ntly actiosphoryled. Two
elative to
signaling
745 was
a to Gly
stent with
ncreasing
in reducpresence
ensitized
d in the
, perhaps
nding by
hain was
Fig. 1. (A to D) In vivo UPR assays using a UPR element– driven lacZ reporter. The relevant
genotypes and the presence or absence of 1NM-PP1 and the UPR inducer DT T are indicated. For
(A) to (C), DT T was used in all assays. wt, wild type; a.u., arbitrary units.
Fig. 2. In vivo HAC1
mRNA splicing and
Hac1 protein production. Northern blots
showing (A) in vivo
HAC1 mRNA splicing
and (B) SCR1 ribosomal RNA (rRNA) as
loading control. Western blots showing (C)
Hac1 and (D) Ire1 protein levels. The arrow in
(C), lane 6, calls attention to the small
amount of Hac1 protein
present in these cells.
requires
1 mRNA
vation by
owed the
1 mRNA
s expectwith DTT
vealed by
o HAC1i
NM-PP1
(Fig. 2A,
th HAC1
lated sig-
PP1–senmRNA
a signifo providalone did
expected,
cing (e.g.,
NM-PP1
xpressing
nt, which
produced
exhibited
roduction
T (Fig. 2,
Ire1 proents, exobserved
ensitized
contrast,
stress.
Thus,thein 1NM-PP1–sensitized
the genetic backgroundIre1
of
(L745A,S840A,S841A)
Ire1(L745G,
1NM-PP1–sensitized
IRE1 and
alleles,
1NM-PP1
is,S840A,S841A)
in effect, a cofactor
for signaling
in the UPR.
mutants
were significantly
1NM-PP1byuncouples
kinase and
activated
1NM-PP1,the
indicating
that
RNase
activities of
phosphorylation
of 1NM-PP1–sensitized
S840 and S841 can be
Ire1. To rule out indirect effects, we assayed
bypassed. Two trends are apparent: With
the activities of the Ire1 mutants described
DTT inalone,
to the portion
parent mutant
above
vitro.relative
The cytosolic
of Ire1
Ire1(S840A,S841A),
decreased
consisting
of the kinasesignaling
domain and
RNase
as the side
chain
residue 745
prodomain
(Ire1*)
wasofexpressed
andwas
purified
(7). Leu
Using
[�-32toP]-lafrom
Escherichia
colifrom
gressively
reduced
to Ala
Gly
beled
we assayed
wild-type
Ire1*
and
(Fig.ATP,
1C, bars
3, 5, and
7). This
is conIre1*(L745G)
for
autophosphorylation
in
the
sistent with the results of Fig. 1A, which
presence or absence of 1NM-PP1. Wild-type
Ire1* exhibited strong autophosphorylation,
6
which
was not inhibited by 1NM-PP1 (Fig. 3A,
lanes 1 and 2). In contrast, Ire1*(L745G) ex-
show an increasing
sensitivity
of thetoacIre1*(L745G)
is diminished
in its ability
use
tive-site
side-chain
at residue
ATP
as to
substrate
and reduction
that 1NM-PP1
is, as
predicted,
an ATP competitor.
745. Reciprocally,
in the presence of 1NMIn contrast,
when
with [�-32P]-laPP1,
activation
of provided
1NM-PP1–sensitized
N-6
benzyl
ATP,
an
ATP
analog with
beled
Ire1(S840A,S841A) mutants increased
in a
bulky substituent (Fig. 3E), Ire1*(L745G) disthe same order (Fig. 1C, bars 4, 6, and 8),
played enhanced autophosphorylation activity
perhapstobecause
of progressively
relative
wild-type
Ire1* (Fig. 3B,enhanced
lane 1 and
binding
by 1NM-PP1
the residue
745
2).
Therefore,
the L745G as
mutation
does not
deside chain
was trimmed
back
stroy
Ire1’s catalytic
function,
but rather alters its
substrate
specificity from ATPIre1
to ATP
analogs
1NM-PP1–sensitized
requires
having
complementary
features
permit
bind1NM-PP1
as a cofactor
for that
HAC1
mRNA
ing
in
the
expanded
active
site.
splicing. We next confirmed that activaTo ask how the RNase activity of Ire1 is
affected by manipulation of its kinase domain,
we incubated wild-type Ire1* and Ire1*(L745G)
with a truncated in vitro transcribed HAC1 RNA
tion by 1NM-PP1–sensitized Ire1 mutants
followed the expected pathway of activating HAC1 mRNA splicing and Hac1 protein production. As expected, UPR induction of wild-type cells with DTT caused
splicing of HAC1 mRNA, as revealed by
near-complete conversion of HAC1u to
HAC1i mRNA (Fig. 2A, lanes 1 and 2) (5);
1NM-PP1 had no effect by itself or with
DTT (Fig. 2A, lanes 3 and 4). In strict correlation with HAC1 mRNA splicing, Hac1
protein accumulated significantly (Fig.
2C, lanes 2 and 4)
In contrast, cells expressing 1NMPP1–sensitized Ire1(L745G) spliced HAC1
mRNA weakly with DTT alone, but displayed a significant increase when 1NMPP1 was also provided (Fig. 2A, lanes 5 to
8). 1NM-PP1 alone did not activate HAC1
mRNA splicing. As expected, Hac1 protein
levels correlated with splicing (e.g., Fig.
2C, lanes 6 and 8). Activation by 1NMPP1 was even more pronounced in cells
expressing the Ire1(L745A,D828A) double mutant, which neither spliced HAC1
mRNA nor produced Hac1 protein with
DTT alone, but exhibited robust splicing
and Hac1 protein production when provided both 1NM-PP1and DTT (Fig. 2, A
and C, compare lanes 10 and 12).
Importantly, steady-state levels of Ire1
proteins were comparable in all experiments, excluding as a trivial explanation
for the observed effects the possibility
that 1NM-PP1–sensitized Ire1 mutants
are only stably expressed in the presence
of 1NM-PP1 (Fig. 2D). Taken together,
our data show that 1NM-PP1 permits but
does not instruct 1NM-PP1–sensitized
Ire1 mutants to splice HAC1 mRNA in response to ER stress. Thus, in the genetic
background of 1NM-PP1–sensitized IRE1
alleles, 1NM-PP1 is, in effect, a cofactor
for signaling in the UPR
1NM-PP1 uncouples the kinase and
RNase activities of 1NM-PP1–sensitized Ire1. To rule out indirect effects, we
assayed the activities of the Ire1 mutants
described above in vitro. The cytosolic
portion of Ire1 consisting of the kinase
domain and RNase domain (Ire1*) was
expressed and purified from Escherichia
coli (7). Using γ-32P]-labeled ATP, we assayed wild-type Ire1* and Ire1*(L745G)
for autophosphorylation in the presence or
absence of 1NM-PP1. Wild-type Ire1* exhibited strong autophosphorylation, which
was not inhibited by 1NM-PP1 (Fig. 3A,
lanes 1 and 2). In contrast, Ire1*(L745G)
exhibited markedly diminished autophosphorylation, consistent with poor signaling by Ire1(L745G) in vivo (Figs. 1 and
2), which was completely extinguished by
1NM-PP1 (Fig. 3A, lane 3 and 4). This
suggested that Ire1*(L745G) is diminished in its ability to use ATP as substrate
and that 1NM-PP1 is, as predicted, an ATP
competitor.
In contrast, when provided with [γ32
P]-labeled N-6 benzyl ATP, an ATP
analog with a bulky substituent (Fig. 3E),
Ire1*(L745G) displayed enhanced autophosphorylation activity relative to wildtype Ire1* (Fig. 3B, lane 1 and 2). Therefore, the L745G mutation does not destroy
Ire1’s catalytic function, but rather alters its
substrate specificity from ATP to ATP analogs having complementary features that
permit binding in the expanded active site.
To ask how the RNase activity of Ire1
is affected by manipulation of its kinase
domain, we incubated wild-type Ire1*
and Ire1*(L745G) with a truncated in
vitro transcribed HAC1 RNA substrate
(HAC1600) and monitored the production
of cleavage products (7). Wild-type Ire1*
cleaved HAC1600 RNA at both splice junctions, producing the expected fragments
corresponding to the singly and doubly cut
species (Fig. 3C, lane 2). The cleavage reaction was not affected by 1NM-PP1 (Fig.
3C, lane 3). In contrast, Ire1*(L745G) was
completely inactive, whereas the addition
of 1NM-PP1, at the same concentration
that was shown in Fig. 3A (lane 4) to completely inhibit the kinase activity, restored
RNase activity significantly (Fig. 3C,
lanes 4 and 5), strongly supporting the no7
RESEARCH A
RESEARCH ARTICLE
Fig. 3. In vitro assays of Ire1 mutant proteins. Ire1* autophosphorylation
32
Fig.
In3.(A)
vitro
assays
of Ire1 of
mutant
proteins.
Ire1* autophos
Fig.
Inand
vitro
Ire1
mutant
assays with [�P]3.ATP
[�-32assays
P]-labeled
N-6
benzyl
ATP (B). Phos32
assays
with(wild-type
[�-32P] ATP
(A) and
P]-labeled
N-6 benzyl AT
phorylated Ire1*
proteins
and L745G)
are[�indicated.
Ire1* RNase
proteins.
Ire1*
autophosphorylation
assays (against
in vitro transcribed
HAC1600
RNA) with and
stimulatory
phorylated
Ire1* proteins
(wild-type
L745G)cofacare indicated. I
32(C) and (D) indicate HAC1
and(against
ADPwith
(D).in
Icons
in
tors 1NM-PP1assays
(C)
P]
ATP
(A)
and[
γassays
[γvitro transcribed HAC1600 RNA)
with stimula
600 RNA
cleavage products,
denoting all(C)
combinations
of
singly
andindoubly
cut RNA.
and
ADP
(D).
Icons
(C)
and
(D)
indicate HA
tors321NM-PP1
P]-labeled
N-6 andbenzyl
(B).
(E) The chemical structures
of 1NM-PP1
N-6 benzylATP
ATP are
shown.
cleavage products, denoting all combinations of singly and doub
Ire1*ofproteins
(E) Phosphorylated
The chemical structures
1NM-PP1 (wildand N-6 benzyl ATP a
Fig. 4. Glycerol-density
expressions
for everyare
mRNA
between the spectype and
L745G)
indicated.
gradient velocity sediified genotypes.
Ire1*
RNase
assays (against
inforvitro
mentation of Fig.
wild-type
4. Glycerol-density
expressions
everyIRE1mRNA betwe
The comparison
of wild-type
and mutant Ire1*.
Ire1* velocity sedigradient
transcribed
HAC1
expressing
cells600with
�ire1
cellswith
revealed a
ifiedRNA)
genotypes.
proteins (A to C) and
mentation
of
wild-type
distinct
set
of
up-regulated
UPR
target
genes
comparison inof wild-ty
Ire1*(L745G) stimulatory
proteins
cofactorsThe 1NM-PP1
mutant wild-type
Ire1*. Ire1*
IRE1-expressing
cells
(Fig.
(D to F) were and
incubated
�ire1 cells
expressing
cellsand
with5A;
(C)
and
ADP
(D).
Icons
in
(C)
to C)thatand
proteins
in the presence
of ADP (A genes
were up-regulated more
than twofold
distinct
setcleavage
of
up-regulated UPR tar
[(B) and (E)] or Ire1*(L745G)
1NM-PP1
are proteins
shown
in 600
pink).
These
IRE1-dependent
(D) indicate
HAC1
RNA
[(C) and (F)] and
subjectwild-type
IRE1-expressing
cells
(Dproducts,
to F) were
incubated
genes
include previously
described
UPR trandenoting
all combinations
ed to velocity in
sedimenthe presence
of ADP
thatNotably,
were up-regulated
more t
scriptional
targetsgenes
(21, 22).
the same
tation analysis.of Sedisingly
and
doubly
cut
RNA.
(E)
The
[(B)
and
(E)]
or
1NM-PP1
set
of
(pink-colored)
UPR
target
genes
was
are
shown
in
pink).
These
IRE1
mentation is from left
[(C)
and (F)] and
subject- to of
up-regulated
a 1NM-PP1
similar
magnitude
chemical
structures
and in described
to right.
genes
include
previously
ed to velocity
sedimencells relative
IRE1(L745A,D828A)-expressing
scriptional targets
(21, 22). Notabl
N-6 benzyl
ATPcells
are (Fig.
shown.
tation
analysis.
to �ire1Sedi5B), suggesting that a caof (pink-colored)
UPR target
mentation
from UPR
left wasset
ent velocity sedimentation. Ire1* sedimented
as isnonical
induced
in the 1NM-PP1–
up-regulated
a similar ma
change
occurs
toconformational
right.
avelocity
broad peak sedimentation
in the gradient (Fig. 4). Upon
the
sensitized, kinase-dead
mutant.
Thistoconclusion
lane 2). The cleavage reaction was not affected
byFig.
1NM-PP1
(Fig. 3C, lane 3). In gradient
contrast,
4. Glycerol-density
c
Ire1*(L745G) was completely inactive, whereas
addition of ADP (Fig. 4B), but not 1NM-PP1
was confirmed byIRE1(L745A,D828A)-expressing
the tight superimposition of
of
wild-type
and
mutant
Ire1*. Ire1*
proteins (A to C)
in response
to cofactor
bindthe addition of 1NM-PP1, at the same concen(Fig. 4C), the peak of Ire1* shifted as a result of
the scatter diagonals
of bothcells
UPR (Fig.
targets5B),
and suggesting
to �ire1
and
Ire1*(L745G)
proteins
(D
to
F)
were
incubated
in
the
tration
that
was
shown
in
Fig.
3A
(lane
4)
to
faster
sedimentation.
Conversely,
the
peak
of
the
rest
of
the
transcriptome
evident
in
the
analyzing
Ire1*
lane 2). The cleavage reaction was not affected
nonical
UPRand
was induced in the
ent velocity sedimentation.ing
Ire1* by
sedimented
as
completely
inhibit
the kinase
Ire1*(L745G)
shifted
a corresponding
of wild-type IRE1- compresence
of (Fig.
ADP
[(B)activity,
and(E)]
1NM-PP1
and(F)]
by
1NM-PP1
3C,
lane
3).restored
Inorcontrast,
kinase-dead mutant. This
a [(C)
broad
peak by
in and
the
gradient
(Fig. 4).expression
Upon theprofilessensitized,
Ire1*(L745G)
glycerolRNase activity significantly (Fig. 3C, lanes 4 and
amount upon the addition of 1NM-PP1 (Fig.
4F)
pared withthrough
IRE1(L745A,D828A)-expressing
subjected
towas
velocity
sedimentation
analysis.
Sedimentation
Ire1*(L745G)
completely
addition
of ADP
(Fig.
but notcells
1NM-PP1
5),
strongly supporting
the notion
thatinactive,
1NM-PP1whereas
but
not upon
the addition
of ADP
(Fig.4B),
4E). This
(Fig. 5C). was confirmed by the tight superim
the
addition
of
1NM-PP1,
at the
sameofconcenthe scatterUPR
diagonals
of both UPR
the peak
Ire1*gradient
shifted as avelocity
result of ansedimentation.
is from
left
to
right.
uncouples
the
kinase
and RNase
activities
shift may(Fig.
be 4C),
indicative
of a of
conformational
Building
instructive
switch.The
tration that was shown
to and/or
restmutants
of peak
thedescribed
transcriptome
evid
fasterchange
sedimentation.
Conversely,
the
peak of asthe
1NM-PP1–sensitized
so
1NM-PP1–sensitized
Ire1. in Fig. 3A (lane 4)change
in the oligomeric
state of sedimented
Ire1*
a IRE1
broad
far act permissively,
because activation
We have previously
shown
that cleavage
the enzymes
in response to shifted
the bindingby
of their
completely
inhibit the
kinase
activity,ofrestored
expression
profilesrequires
of wild-type I
Ire1*(L745G)
a corresponding
in the gradient (Fig.
4).
Uponpared
the
addition
tionactivity
that
1NM-PP1
the kinase
both
1NM-PP1
protein-misfolding
agents.
HAC1
by significantly
wild-type
Ire1*
isuncouples
stimulated
cofactors.
RNasemRNA
(Fig.
3C, lanes 4 respective
and
with IRE1(L745A,D828A)
amount
upon the addition of 1NM-PP1
(Fig.
4F) and
1NM-PP1–sensitized,
kinase-dead
Ire1
This
indicates
that an unfolded-protein
(ADP) (7).
This
is
by adenosine
5�-diphosphate
of addition
ADP
(Fig.
4B),
but
not 1NM-PP1
(Fig. signal
RNase
activities
1NM-PP1–sensi5), and
strongly
supporting
the
notionof
that
1NM-PP1
cells (Fig. 5C).
but not upon the
of ADP
(Fig.
4E).
This
signals a canonical UPR. To ask whether
needs to be received by the ER lumenal domain
consistent with the notion derived from in vivo
uncouples
the requires
kinase an
andactive
RNase
of
anof
instructive
UPR
shift
maykinase
be4C),
indicative
of1NMa of
conformational
bypassing
the Ire1
activity
for activation.
We asked
we could
bypass
this
studies
that Ire1
kinaseactivities
dothewith
peak
Ire1*
shifted
asBuilding
aifresult
tized
Ire1.
IRE1
1NM-PP1–sensitized
Ire1.
change
and/or change
the oligomeric
state ofusing1NM-PP1–sensitized
PP1 allows
the induction
of a fullin UPR,
we
requirement
a constitutively on version
of mutants d
main,
because we know that
recombinant wildfaster
sedimentation.
Conversely,
peak through
We
have
previously
shown
that
cleavC
act the
permissively,
because activat
have
previously
shown thatthereby
cleavageprofiled
of mRNA
the
enzymes
in response
to
the binding
theirIRE1far
) isolated
previously
expression
on yeast
genomic
IRE1of(called
type We
Ire1*
is already
phosphorylated,
random
mutagenesis
of the
ER lumenal
microarrays.
To this cofactors.
end,
from wildalleviating
a necessity
for de
novoIre1*
phosphoryl1NM-PP1
anddomain
protein-misfold
HAC1
by wild-type
is stimulated
respective
of mRNA
Ire1*(L745G)
shifted
byC aboth
correspondagemRNA
of
HAC1
mRNA
by
wild-type
Ire1*
significantly
induced
the
UPR
(18,
21).
IRE1
type
IRE1,
�ire1,
and
IRE1(L745A,D828A)
ation
in
vitro.
ADP
efficiently
stimulated
the
1NM-PP1–sensitized, kinase-dead Ire1
This indicates that an
unfolded-pr
by adenosine 5�-diphosphate (ADP) (7). This is
is
stimulated
by
adenosine
ing
amount
addition
of
1NM-PP1
without
DTT, resulting
in about 75% of the
cells was isolated at different
points in upon
time the
RNase
activity
of Ire1* (Fig.
3D,
lane 1) but not5′-diphosphate
signals a canonical UPR. To ask whether
needs to be received by the ER lume
consistent with the notion derived from in vivo
maximal activity of wild-type IRE1 with DTT
after the addition of 1NM-PP1 and DTT. cDthat of Ire1*(L745G) (Fig. 3D, lane 2).
4F)
but
not upon
the
of ADP
(ADP)
(7).
This
isanconsistent
the
nobypassing
the(Fig.
Ire1
kinase
activity
with
1NMWe asked
studies
that Ire1
active kinase with
do- derived
(Fig. 1D,
barsaddition
2 for
and activation.
5). We
predicted
that aif we could
NAs
from these
mRNA
populations
For
wild-type
Ire1requires
and 1NM-PP1–sensitized
C
PP1
allows
the
induction
of
a full
UPR,
requirement
using
main,
because
thatinrecombinant
wildand 1NM-PP1–sensitized,
chimera
ofwe
thebe
IRE1
were that
labeled
with
different
fluorescent
dyes,
Ire1(L745G),
ADPwe
andknow
1NM-PP1,
respectively,
(Fig.
4E). This
shift
may
indicative
ofCa constitutively o
tion
derived
from
vivo studies
Ire1
) isolated previou
profiledandmRNA
expression
on yeast
genomic IRE1(L745A,D828A)
IRE1 (called IRE1 mutants
type Ire1*
is already
phosphorylated,
kinase-dead
mixed pairwise,
hybridized
to microarrays
function
as stimulatory
cofactors.
Therefore, wethereby
aToconformational
change
and/or
change
requires
an active
domain,
because
would
activate the
UPR with
1NM-PP1 alone,
(20). Scatter
plots of pairwise
comparisons
of
asked
whethera anecessity
conformational
change
random
mutagenesis
of the ER lume
microarrays.
this
end, mRNA
from
wildalleviating
for dekinase
novooccurs
phosphorylC in Fig. 1D
even in the absence(18,
of DTT.
The
data
mRNA
expression
levels
areandshown
in
in
response
to cofactor
binding
by analyzing
significantly
induce
21).
IRE1
type
IRE1,
�ire1,
IRE1(L745A,D828A)
ation
in
vitro.
ADP
efficiently
stimulated
the
state
of the enzymes in
we know that recombinant wild-type
Ire1* in the oligomeric(bars
13 to 16) show that this prediction holds
Fig. 5; the x-y scatter plots represent relative
Ire1* and Ire1*(L745G) through glycerol-gradi-
without DTT, resulting in about 7
cells was isolated at different points in time
RNase activity of Ire1* (Fig. 3D, lane 1) but not
response to the binding of their
respective
is already phosphorylated, thereby allevimaximal activity of wild-type IRE1
after the addition of 1NM-PP1 and DTT. cDthat of Ire1*(L745G) (Fig. 3D, lane 2).
www.sciencemag.org SCIENCE VOL 302 28 NOVEMBER 2003
1535
ating
a necessity
for de novo phosphory(Fig. 1D, bars 2 and 5). We pred
NAs derivedcofactors.
from these mRNA populations
For wild-type
Ire1 and 1NM-PP1–sensitized
C
and
1NM-PP1
chimera
of
the
IRE1
were
labeled
with
different
fluorescent
dyes,
Ire1(L745G),
ADP
and
1NM-PP1,
respectively,
1NM-PP1–sensitized, kinase-dead
lation in vitro. ADP efficiently stimulated
kinase-dead IRE1(L745A,D828A
mixed pairwise, and hybridized to microarrays
function as stimulatory cofactors. Therefore, we
a canonical
To ask
thewhether
RNase
activity of Ire1*
lane
would activate
the UPR with 1NM
(20).
Scatter Ire1
plots ofsignals
pairwise comparisons
of UPR.
asked
a conformational
change (Fig.
occurs 3D,
even in the
absence of DTT. The dat
mRNA
are shown
in kinase
in 1)
response
to cofactor
by analyzing(Fig.
whetherlevels
bypassing
the Ire1
activity
but not
that ofbinding
Ire1*(L745G)
3D, expression
(bars 13 to 16) show that this pred
Fig. 5; the x-y scatter plots represent relative
Ire1* and Ire1*(L745G) through glycerol-gradi-
lane 2).
For wild-type Ire1 and 1NM-PP1–senwww.sciencemag.org
sitized Ire1(L745G), ADP and 1NM-PP1,
respectively, function as stimulatory cofactors. Therefore, we asked whether a
8
with 1NM-PP1 allows the induction of a
full
UPR,VOL
we302
profiled
mRNA2003
expression
SCIENCE
28 NOVEMBER
on yeast genomic microarrays. To this end,
mRNA from wildtype IRE1, Δire1, and
IRE1(L745A,D828A) cells was isolated
1NM-PP1 alone (i.e., even in the absence of
ER stress).
identified. Here, we report that both the
kinase activity and activation-segment phos-
Fig. 5. (A to C) x-y scatter plot analysis of mRNA abundance. The plots (log10) compare yeast
genomic microarray analyses between the genotypes indicated. In each case, mRNA was purified
from cells that were provided both DT T and 1NM-PP1. Pink dots represent genes with expression
more than two times as high in wild-type cells as in �ire1 cells.
Fig. 6. Model of activation of 1NM-PP1–sensitized and wild-type Ire1. For both proteins, chaperone
dissociation resulting from unfolded-protein accumulation causes oligomerization in the plane of
the ER membrane, which is a precondition for further activation. For mutant Ire1 (A), 1NM-PP1
binding to the inactive kinase domain active site causes a conformational change, which activates
the RNase. For wild-type Ire1 (B), trans-autophosphorylation, with ATP, of the activation segment
fully opens the kinase domain for binding of ADP or ATP, which activates the RNase.
diated by
RNase a
Ire1, th
mechani
One m
proposed
model,
Ire1 occ
after the
chaperon
by latera
membra
ation of
activatio
(24, 25 )
allowing
leading
that acti
In co
the activ
occurs e
ation of
sible tha
PP1 can
ment, w
to the a
ATP mo
1NM-PP
the unp
opens tr
frequenc
rate of
sensitize
ADP and
could b
Based o
drug bin
rearrang
the RNa
phospho
mimics
phospho
The m
why 1NM
1NM-PP1
vate them
by activa
nal doma
the obser
may be e
free muta
well to c
(or cons
may be
neighbori
formation
labeled
withVOL
different
fluorescentwww.sciencemag.org
dyes,
1536 at different points in time after the
28addiNOVEMBER
2003
302 SCIENCE
tion of 1NM-PP1 and DTT. cDNAs derived from these mRNA populations were
mixed pairwise, and hybridized to microarrays (20). Scatter plots of pairwise
9
comparisons of mRNA expression levels
are shown in Fig. 5; the x-y scatter plots
represent relative expressions for every
mRNA between the specified genotypes.
The comparison of wild-type IRE1expressing cells with Δire1 cells revealed
a distinct set of up-regulated UPR target
genes in wild-type IRE1-expressing cells
(Fig. 5A; genes that were up-regulated
more than twofold are shown in pink).
These IRE1-dependent genes include
previously described UPR transcriptional
targets (21, 22). Notably, the same set
of (pink-colored) UPR target genes was
up-regulated to a similar magnitude in
IRE1(L745A,D828A)-expressing
cells
relative to Δire1 cells (Fig. 5B), suggesting that a canonical UPR was induced in
the 1NM-PP1–sensitized, kinase-dead
mutant. This conclusion was confirmed
by the tight superimposition of the scatter
diagonals of both UPR targets and the rest
of the transcriptome evident in the expression profiles of wild-type IRE1- compared
with
IRE1(L745A,D828A)-expressing
cells (Fig. 5C).
Building an instructive UPR switch.
The 1NM-PP1–sensitized IRE1 mutants
described so far act permissively, because activation requires both 1NM-PP1
and protein-misfolding agents. This indicates that an unfolded-protein signal
needs to be received by the ER lumenal
domain for activation. We asked if we
could bypass this requirement using a
constitutively on version of IRE1 (called
IRE1C) isolated previously through random mutagenesis of the ER lumenal domain (18, 21). IRE1C significantly induced
the UPR without DTT, resulting in about
75% of the maximal activity of wild-type
IRE1 with DTT (Fig. 1D, bars 2 and 5).
We predicted that a chimera of the IRE1C
and 1NM-PP1–sensitized, kinase-dead
IRE1(L745A,D828A) mutants would activate the UPR with 1NM-PP1 alone, even
in the absence of DTT. The data in Fig. 1D
(bars 13 to 16) show that this prediction
holds true. Whereas wild-type IRE1 re10
quires DTT for induction and is immune
to 1NM-PP1 (Fig. 1D, bars 1 to 4), and
IRE1(L745A,D828A) requires both DTT
and 1NM-PP1 (Fig. 1D, bars 9 to 12), the
chimeric IRE1C (L745A,D828A) requires
only 1NM-PP1 for activation, irrespective
of the addition of DTT (Fig. 1D, bars 13
to 16). Thus, IRE1C (L745A,D828A) is an
instructive switch that turns on the UPR
upon the addition of 1NM-PP1 alone (i.e.,
even in the absence of ER stress).
Summary and implications. Since
our discovery that Ire1’s RNase initiates
splicing of HAC1 mRNA, the function of
its kinase domain has remained an enigma
(7). Although mutational analyses have
indicated a requirement for an enzymatically active kinase and have defined activation-segment phosphorylation sites (9),
to date no targets besides Ire1 itself have
been identified. Here, we report that both
the kinase activity and activation-segment
phosphorylation can be bypassed if the
small ATP-mimic 1NM-PP1 is provided to
a mutant enzyme to which it can bind. Instead of inhibiting the function served by
ATP, 1NM-PP1 rectifies the signaling defect of 1NM-PP1–sensitized Ire1 mutants,
leading to the induction of a canonical
UPR (21). Thus, ligand binding to the kinase domain, rather than a phosphotransfer function mediated by the kinase activity, is required for RNase activation. The
kinase module of Ire1, therefore, uses an
unprecedented mechanism to propagate
the UPR signal.
One model that could explain our data
is proposed in Fig. 6B. According to this
model, autophosphorylation of wild-type
Ire1 occurs in trans between Ire1 molecules after they have been released from
their chaperone anchors and have oligomerized by lateral diffusion in the plane of
the ER membrane (9, 23). Trans-autophosphorylation of the activation segment locks
the activation segment in the “swung-out”
state (24, 25), and fully opens the active
site, allowing ADP or ATP to bind efficiently, leading in turn to conformational
changes that activate the RNase domain.
In contrast, the binding of 1NM-PP1
to the active site of 1NM-PP1–sensitized
Ire1 occurs even in the absence of phosphorylation of the activation segment. It
is plausible that because of its small size,
1NMPP1 can bypass the “closed” activation segment, which is proposed to occlude
access to the active site for the larger ADP
and ATP molecules (Fig. 6A). Alternatively, 1NM-PP1 may enter the active site
when the unphosphorylated activation segment opens transiently, which occurs with
low frequency in other kinases (26). If the
offrate of 1NM-PP1 binding to 1NM-PP1–
sensitized Ire1 is slow compared to that of
ADP and ATP, most mutant Ire1 molecules
could be trapped in a drug-bound state.
Based on either scenario, we propose that
drug binding alone causes conformational
rearrangements in the kinase that activate
the RNase. The nucleotide-bound state of
phosphorylated wild-type Ire1, therefore,
mimics the 1NM-PP1–bound state of unphosphorylated 1NM-PP1–sensitized Ire1.
The model discussed so far does not
explain why 1NM-PP1 should not bind
to individual 1NM-PP1–sensitized Ire1
molecules and activate them, even without
oligomerization induced by activation of
function.
Currently,
not allow
us to
the UPR
throughour
thedata
ERdo
lumenal
domain.
distinguish between these possibilities.
Two
couldthe
account
forofthe
obOurpossibilities
findings provoke
question
what
servations:
(i) The binding
the
“natural” stimulatory
ligandof
of 1NM-PP1
Ire1’s kimaydomain
be enhanced
chapnase
may be. in
Oneoligomerized,
likely candidate
is
ADP,
which mutants,
is generated
by Ire1
from ATP
erone-free
or (ii)
1NM-PP1
may
during
the autophosphorylation
reaction. In
bind equally
well to chaperone-anchored
vitro,
is a better activator
of Ire1 than
and ADP
chaperone-free
(or constitutive-on)
ATP (7). Ire1 would therefore be “self primIre1,generating
but oligomerization
may be required
ing,”
an efficient activator
already
because
neighboring
bound
to itsinteraction
active site. between
In many professional
molecules
is required
for �-cells
the conformasecretory
cells
such as the
of the
endocrine
pancreas
contain
high funccontional change
that (which
activates
the RNase
centrations
of Ire1),
ADPdolevels
rise (and
tion. Currently,
our data
not allow
us to
ATP levels decline) temporarily in proportion
distinguish between these possibilities.
to nutritional stress (27). ADP therefore is
Our findings
provoke
theasquestion
physiologically
poised
to serve
a cofactorof
what
the
“natural”
stimulatory
ligand
that could signal a starvation state.
It isof
Ire1’s that
kinase
domain
maybecomes
be. Oneineffilikely
known
protein
folding
cient
as the nutritional
status of
declines,
candidate
is ADP, which
is cells
generated
by
triggering
UPR
(28). the
Thus,
in the face of
Ire1 fromtheATP
during
autophosphoryATP depletion, ADP-mediated conformalation reaction. In vitro, ADP is a better
tional changes might increase the dwell time
of activated Ire1. As such, Ire1 could have
evolved this regulatory mechanism to monitor the energy balance of the cell and couple
activator of Ire1 than ATP (7). Ire1 would
therefore be “self priming,” generating an
efficient activator already bound to its active site. In many professional secretory
cells such as the β-cells of the endocrine
pancreas (which contain high concentrations of Ire1), ADP levels rise (and ATP
levels decline) temporarily in proportion
to nutritional stress (27). ADP therefore
is physiologically poised to serve as a cofactor that could signal a starvation state.
It is known that protein folding becomes
inefficient as the nutritional status of cells
declines, triggering the UPR (28). Thus, in
the face of ATP depletion, ADP-mediated
conformational changes might increase
the dwell time of activated Ire1. As such,
Ire1 could have evolved this regulatory
mechanism to monitor the energy balance
of the cell and couple this information to
activation of the UPR.
Although unprecedented in proteins
containing active kinase domains, our
findings are consistent with previous observations of RNase L, which is closely
related to Ire1, bearing a kinase-like domain followed C-terminally by an RNase
domain (29, 30). Similar to Ire1, RNase
L is activated by dimerization (31). However, RNase L’s kinase domain is naturally
Similar
Ire1,whereas
RNaseitsL RNase
is activated
inactiveto(29),
activitybyis
dimerization (31). However, RNase L’s kistimulated
bynaturally
adenosine
nucleotide
bindnase
domain is
inactive
(29), wherethe kinase
Therefore,
like
asing
its to
RNase
activitydomain.
is stimulated
by adenoIre1,nucleotide
the ligand-occupied
domain
sine
binding to the kinase domain.
Therefore,
Ire1, the
kiof RNaselike
L serves
as ligand-occupied
a module that parnase
domain
of RNase Land
serves
as a module
ticipates
in activation
regulation
of the
that
participates
activation
and regulation
RNase
function.inInsights
gained
from Ire1
of the RNase function. Insights gained from
and
RNase
L
may
extend
to
other
proteins
Ire1 and RNase L may extend to other
procontaining
kinase
or or
enzymatically
teins
containing
kinase
enzymaticallyinacintive pseudokinase
(32).
active
pseudokinase domains
domains (32).
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l transmission
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1 Doppler effect
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*
reproducible manner
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exciting
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1999).
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Theoretical
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Chapel
Hill,more
NC 27599,
USA.
National
Center
for
Oxford,
1981), ch.
6.
clude
laser
flow
14)
and
use
of
shock
discontinuities
in
Biotechnology
National
Institutes
ofonHealth,
we
focus
the
be
problematic
synonymous
polymorphisms
have
been
widegrading
ca
refers
to frequency
shifts
areblood
inThe
the
opddressed.
clude
radar,
laser
vibrometry,
blood
flow
14)
and the
theInformation,
use(2).
of Here,
shock
discontinuities
in
molecular
basis
of vibrometry,
human
disease.
ef5. P. radar,
Bayliss,
D.
Choquenot,
Proc.that
R.
Soc.
London
Ser.
B
ddressed.
E(Univ. of E3
Thurston
ArthritisofCenter,
Bethesda, MD
20894,(15)
USA.
measurement,
and
the
search
for
astrophotonic
crystals
and
transmission
lines
357,sense
1233 (2002).
whereby
polymorphisms
the
ly
characterized;
because
these
variations
critical com
posite
to
described
above;
for mechanism
measurement,
andthose
the
search
for new
new
astrophotonic
crystals
(15)
and
transmission
lines
fects
of
nonsynonymous
polymorphisms
University
of
North
Carolina,
Chapel
Hill,
NC
27599,
USA.
6. C. G. Jones,
R. S. Ostfeld,
M. P. Richard, E.would
M. Schauber,
ntal Care
4
(COMT
) gene
(3). The
example,
increased
frequencies
be cathechol-O-methyltransferase
Attagene, Inc., 7030 Kit Creek Road, Research
Triangle
Park,
have
characterized;
because
J. been
O. Wolff,widely
Science 279,
1023 (1998).
9).
1measured on reflection of waves from a reregulate
distinct pr
NC2003
27560,gene
USA. expression.
www.sciencemag.org
SCIENCE
VOL
302
28
NOVEMBER
2003
1537
Center
for
Neurosensory
Disorders,
University
of
North
7.
R.
S.
Ostfeld,
F.
Keesing,
Trends
Ecol.
Evol.
15,
232
(2000).
www.sciencemag.org
SCIENCE
VOL
302
28
NOVEMBER
1537
May, Ed.
these
variations
directly
influence
protein
2
is an enzyme
responsible
forE-mail:
deand mem
Carolina,
Chapel
NC Annu.
27599,
USA.
Center
for
ceding
boundary.
Demonstration
8. D. Kelly,
V. Hill,
L. Sork,
Rev.
Ecol.National
Syst. 33,of
427this
*ToCOMT
whom correspondence
should
be addressed.
Biotechnology
Information,
National Institutes
function,
are
relatively
easy toof study
catecholamines and thus represents a
through th
(2002). they
[email protected]
counterintuitive
phenomenon
requires
aHealth,
fun- grading
ndon Ser. B
3
Bethesda, MD 20894, USA. Thurston Arthritis Center,
the
damental
change
in theChapel
wayHill,
thatNCradiation
is critical component of homeostasis maintenance
University
of
North
Carolina,
27599,
USA.
. M. Schauber,
4reflected from a moving boundary, and, de(3). The human COMT gene encodes two
This
12
Attagene, Inc., 7030 Kit Creek Road, Research Triangle Park,
1930NC
22 DECEMBER
2006proteins:
VOL 314
www.sciencemag.org
distinct
solubleSCIENCE
COMT (S-COMT)
27560,
USA. range of theoretical work that
ving
spite
a wide
15, 232 (2000).
and membrane-bound COMT (MB-COMT)
R EPORTS
tion of the Inverse
oppler Effect
Human Catechol-O-Methy
Haplotypes Modulate Prot
by Altering mRNA Secon
Human Catechol-O-Methyltransferase
Modulate Protein Expression
tion ofHaplotypes
the Inverse
Altering mRNA Secondary Structure
oppler by
Effect
R EPORTS
oppler Effect
RTTEPORTS
T
AAcommon
commonSNP
SNPinincodon
codon158
158(val
(val met)
notype.Because
Becausevariation
variationininthetheS-COMT
S-COMT forforthethes
met) notype.
structur
promoterregion
regiondoes
doesnot
notcontribute
contributetotopain
pain structure,
has
hasbeen
beenassociated
associatedwith
withpain
painratings
ratingsand
and promoter
longest,
longestm
ininthetheva
MB-COM
MB-CO
Fig.
Fig.1.1.Common
Commonhaplohaploloop
loop
struc
stru
types
types
ofof
thethe
human
human
COMT
COMT
~17
~17kcal/m
kca
gene
genediffer
differwith
withrespect
respect
LPS
LPS
haplo
hap
toto
mRNA
mRNA
secondary
secondary
strucstruc(Fig.
(Fig.
1B1B
a
ture
tureand
andenzymatic
enzymaticac-acporting
portingp
tivity.
tivity.(A)(A)A Aschematic
schematic
obtained
obtained
diagram
diagramillustrates
illustratesCOMT
COMT
ondary
ondarystr
genomic
genomic
organization
organization
and
and
ofofCOMT
COM
SNP
SNPcomposition
compositionforforthethe
three
threehaplotypes.
haplotypes.PerPerspecies
species(f
cent
centfrequency
frequencyofofeach
each
structures
structur
haplotype
haplotypeinina cohort
a cohortofof
highly
highlysta
healthy
healthyCaucasian
Caucasianfe-fegous
goustotot
males,
males,and
andpercent
percentindeindestantial
stantiald
pendent
pendent
SNP
SNP
contribution
contribution
observed
observe
totopain
painsensitivity,
sensitivity,areare
notable
notablef
indicated.
indicated.(B)(B)The
Thelocal
local
studies
studiesw
stem-loop
stem-loopstructures
structuresas-asmodeling
modelin
sociated
sociatedwith
witheach
eachofof
We
We
coc
thethethree
threehaplotypes
haplotypesareare
cDNA
cDNAcl
shown.
shown.Relative
Relativetotothethe
tors
torsthat
th
LPS
LPSand
andAPS
APShaplotypes,
haplotypes,
correspon
corresp
thetheHPS
HPSlocal
localstem-loop
stem-loop
lotypes
lotypes(
structure
structurehad
hada ahigher
higher
were
weretran
tr
folding
foldingpotential.
potential.(C(Cand
and
sixsixconstr
con
D)D)
The
The
LPS
LPS
haplotype
haplotype
ex-extein
teinexpr
ex
hibited
hibited
thethe
highest,
highest,
while
while
measured
measure
thethe
HPS
HPS
haplotype
haplotype
exhibexhibHPS
HPShap
h
ited
ited
thethe
lowest
lowest
enzymatic
enzymatic
reduction
reductio
activity
activityand
andprotein
proteinlevlevelselsinincells
cellsexpressing
expressing
MB-COM
MB-CO
COMT.
COMT.***P
***P< <0.001,
0.001,≠ ≠
and
andfig.
figS
+++
+++
LPS.
LPS. P <
P<
0.001,
0.001,
≠≠
APS.
APS.
ited
itedmark
ma
protein
proteinex
APS
APS
hapl
ha
fold
foldredu
red
MB-COM
MB-CO
three
ed in regulatory regions, whichwww.sciencemag.org
are
much group demonstrated
www.sciencemag.org
SCIENCE
SCIENCEthat
VOL
VOL
314
314common
2222DECEMBER
DECEMBER200
20
more common, has proved to be problem- haplotypes of the human COMT gene are
atic (2). Here, we focus on the mechanism associated with pain sensitivity and the
whereby polymorphisms of the cathechol- likelihood of developing temporomanO-methyltransferase (COMT) gene regu- dibular joint disorder (TMJD), a common
chronic musculoskeletal pain condition
late gene expression.
COMT is an enzyme responsible (4). Three major haplotypes are formed
for degrading catecholamines and thus by four single-nucleotide polymorphisms
represents a critical component of ho- (SNPs): one located in the S-COMT promeostasis maintenance (3). The human moter region (A/G; rs6269) and three in
COMT gene encodes two distinct pro- the S- and MB-COMT coding region at
teins: soluble COMT (S-COMT) and codons his62his (C/T; rs4633), leu136leu (C/
membrane-bound COMT (MB-COMT) G; rs4818), and val158met (A/G; rs4680)
through the use of alternative translation (Fig. 1A). On the basis of subjects’ pain
initiation sites and promoters (3). Re- responsiveness, haplotypes were designatcently, COMT has been implicated in the ed as low (LPS; GCGG), average (APS;
modulation of persistent pain (4–7). Our ATCA), or high (HPS; ACCG) pain sen13
ture and
and converts
converts itit into
into aa LPS
LPS haplotype–like
haplotype–like
ture
structure (HPS
(HPS Lsm).
Lsm). Double
Double mutation
mutation of
of
structure
Fig. 2.
2. Site-directed
Site-directedmumuFig.
tagenesis that
that destroys
destroys
tagenesis
thestable
stablestem-loop
stem-loopstrucstructhe
turecorresponding
correspondingtotothe
the
ture
HPS haplotype
haplotype restores
restores
HPS
COMT enzymatic
enzymatic activity
activity
COMT
and protein
protein expression.
expression.
and
(A) The
The mRNA
mRNA structure
structure
(A)
correspondingtotothe
theHPS
HPS
corresponding
haplotype was
was converted
converted
haplotype
an LPS
LPS haplotype-like
haplotype-like
toto an
structure (HPS
(HPS Lsm)
Lsm) by
by
structure
single mutation
mutation ofof 403C
403C
single
G. The
The original
original HPS
HPS
toto G.
haplotype structure
structure (HPS
(HPS
haplotype
dm)was
wasrestored
restoredby
bydoudoudm)
ble mutation
mutation ofof interactinteractble
ingnucleotides
nucleotides403C
403CtotoGG
ing
and479G
479GtotoC.C.(B
(Band
andC)
C)
and
The HPS
HPS Lsm
Lsm exhibited
exhibited
The
COMT enzymatic
enzymatic activity
activity
COMT
and protein
protein levels
levels equivequivand
alent toto those
those ofof the
the LPS
LPS
alent
haplotype, whereas
whereas the
the
haplotype,
HPS dm
dm exhibited
exhibited rereHPS
duced enzymatic
enzymatic activity.
activity.
duced
***P<<0.001,
0.001,≠≠LPS.
LPS.
***P
verified by
by an
an alternate
alternate approach
approach of
of modifying
modifying
verified
mRNA secondary
secondary structure
structure (fig.
(fig. S5).
S5).
mRNA
Refer
Refere
Y
1.1. L.L.Y.Y.Ya
Hum.M
Hum.
K
2.2. J.J.C.C.Kn
3.3. P.P.T.T.MM
(1999
(1999)
Dia
4.4. L.L. Diat
(2005
(2005)
5.
J.
J.
M
5. J. J. Ma
(1976
(1976)
R
6.6. T.T.T.T.Ra
Z
7.7. J.J.K.K.Zu
Win
8.8. G.G.Win
134(2(2
134
Fun
9.9. B.B.Funk
10. G.G.Oros
Oro
10.
(2004
(2004)
11. J.J.Duan
Dua
11.
(2003
(2003)
12. L.L.X.X.Sh
S
12.
Acad.S
Acad.
13. I.I.Puga
Pug
13.
14. K.K.Mita
Mit
14.
203,99
203,
15. T.T.D.
D.SS
15.
H.J.J.Le
L
H.
(1994
(1994)
16. A.A.Shal
Sha
16.
(2002
(2002)
17.
Materi
17. Materia
materi
materia
18. D.
D.H.
H.M
18.
Biol.22
Biol.
19. M.
M.Zuk
Zuk
19.
20. A.A.Y.Y.OO
20.
M.A.A.R
M.
21. Sequen
Seque
21.
CA489
CA4894
haplot
haploty
threeh
three
sitewe
we
site
enzym
enzyme
threeS
three
throug
through
(BI835
(BI835
endsasa
ends
22. S.S.A.A.Sh
S
22.
AcidsR
Acids
sitive. Individuals
carrying HPS/APS
or APS/APS diplotypes were nearly
2.5 times as likely
to develop TMJD.
Previous data further suggest that
1932 COMT haplotypes code for differences
22 DECEMBER
DECEMBER
2006 VOL
VOL 314
314 associations
SCIENCE between
www.sciencemag.o
1932
22
2006
SCIENCE
www.sciencemag.or
in However,
observed
COMT enzymatic activity (4); however, these conditions and the met allele are ofthe molecular mechanisms involved have ten modest and occasionally inconsistent
remained unknown.
(3). This suggests that additional SNPs in
A common SNP in codon 158 (val158met) the COMT gene modulate COMT activhas been associated with pain ratings and ity. Indeed, we found that haplotype rather
μ-opioid system responses (7) as well as than an individual SNP better accounts for
addiction, cognition, and common affec- variability in pain sensitivity (4). The HPS
tive disorders (3, 8–10). The substitution and LPS haplotypes that both code for the
of valine (Val) by methionine (Met) results stable val158 variant were associated with
in reduced thermostability and activity of the two extreme-pain phenotypes; thus,
the enzyme (3). It is generally accepted the effect of haplotype on pain sensitivthat val158met is the main source of indi- ity in our study cannot be explained by
vidual variation in human COMT activity; the sum of the effects of functional SNPs.
numerous studies have identified associa- Instead, the val158met SNP interacts with
tions between the low-activity met allele other SNPs to determine phenotype. Beand several complex phenotypes (3, 8, 10). cause variation in the S-COMT promoter
14
region does not contribute to pain phenotype (Fig. 1A), we suggest that the rate of
mRNA degradation or protein synthesis
is affected by the structural properties of
the haplotypes, such as haplotype-specific
mRNA secondary structure.
Previous reports have shown that
polymorphic alleles can markedly affect
mRNA secondary structure (11, 12), which
can then have functional consequences on
the rate of mRNA degradation (11, 13).
It is also plausible that polymorphic alleles directly modulate protein translation
through alterations in mRNA secondary
structure, because protein translation efficiency is affected by mRNA secondary
structure (14–16). To test these possibilities, we evaluated the effect of LPS, APS,
and HPS haplotypes on the stability of the
corresponding mRNA secondary structures (17).
Secondary structures of the full-length
LPS, APS, and HPS mRNA transcripts
were predicted by means of the RNA
Mfold (18, 19) and Afold (20) programs.
The mRNA folding analyses demonstrated that the major COMT haplotypes
differ with respect to mRNA secondary
structure. The LPS haplotype codes for
the shortest, least stable local stem-loop
structure, and the HPS haplotype codes
for the longest, most stable local stemloop structure in the val158 region for both
S-COMT and MB-COMT. Gibbs free energy (ΔG) for the stem-loop structure associated with the HPS haplotype is ~17
kcal/mol less than that associated with
the LPS haplotype for both S-COMT and
MB-COMT (Fig. 1B and fig. S1A). Additional evidence supporting predicted
RNA folding structures was obtained by
generating consensus RNA secondary
structures based on comparative analysis
of COMT sequences from eight mammalian species (fig. S2). The consensus RNA
folding structures were LPS-like and did
not contain highly stable local stem-loop
structures analogous to the human HPSlike form. Thus, substantial deviation
from consensus structure, as observed for
the HPS haplotype, should have notable
functional consequences. Additional studies were conducted to test this molecular
modeling.
We constructed full-length S- and MBCOMT cDNA clones in mammalian expression vectors that differed only in three
nucleotides corresponding to the LPS, APS,
and HPS haplotypes (17, 21). Rat adrenal
(PC-12) cells were transiently transfected
with each of these six constructs. COMT
enzymatic activity, protein expression, and
mRNA abundance were measured. Relative to the LPS haplotype, the HPS haplotype showed a 25- and 18-fold reduction in
enzymatic activity for S- and MB-COMT
constructs, respectively (Fig. 1C and fig.
S1B). The HPS haplotype also exhibited
marked reductions in S- and MB-COMT
protein expression (Fig. 1D and fig. S1C).
The APS haplotype displayed a moderate
2.5- and 3-fold reduction in enzymatic
activity for S- and MB-COMT constructs,
respectively, while protein expression levels did not differ. The moderate reduction
in enzymatic activity produced by the APS
haplotype is most likely due to the previously reported decrease in protein thermostability coded by the met158 allele (3).
These data illustrate that the reduced enzymatic activity corresponding to the HPS
haplotype is paralleled by reduced protein
levels, an effect that could be mediated by
local mRNA secondary structure at the level of protein synthesis and/or mRNA degradation. Because total RNA abundance
and RNA degradation rates did not parallel
COMT protein levels (fig. S2), differences
in protein translation efficiency likely results from differences in the local secondary structure of corresponding mRNAs
To directly assess this hypothesis, we
performed site-directed mutagenesis (17).
The stable stem-loop structure of S- and
MB-COMT mRNA corresponding to the
HPS haplotype is supported by base pairs
between several critical nucleotides, including 403C and 479G in S-COMT and
15
in thermo(3). These
tic activity
paralleled
at could be
structure at
or mRNA
ndance and
lel COMT
in protein
om differre of cor-
hesis, we
esis (17).
of S- and
o the HPS
s between
ing 403C
d 701G in
Mutation
C to G in
oop strucotype–like
utation of
structure (HPS dm). The single- and doublenucleotide HPS mutants (HPS Lsm and HPS
dm, respectively) were transiently transfected to
PC-12 cells. As predicted by the mRNA
secondary-structure folding analyses, the HPS
625Cexhibited
and 701G
in MB-COMT
(Fig. 2A
Lsm
increased
COMT enzymatic
and fig.
of 403Cto to
G
activity
andS3A).
proteinMutation
levels equivalent
those
ofin the
LPS haplotype,
HPS dm
S-COMT
or 625C whereas
to G in the
MB-COMT
exhibited
enzymatic
activity structure
and prodestroys reduced
the stable
stem-loop
tein
equivalent
thosehaplotype–like
of the original
andlevels
converts
it into to
a LPS
HPS haplotype (Fig. 2, B and C; fig. S3, B and
structure
(HPS
mutation
of
C).
These data
ruleLsm).
out theDouble
involvement
of RNA
mRNA in
position 403C
and 479G
sequence
recognition
motifs to
or Gcodon
usage to
in
C in
S-COMT
625C toInG contrast
and 701G
to
the
regulation
of or
translation.
to the
HPS
protein
levels didthe
notoriginal
parallel
C inhaplotype,
MB-COMT
reconstructs
COMT
enzymatic activity
for the
APSdm).
haplotype
long stem-loop
structure
(HPS
The
and site-directed mutagenesis confirmed that the
singleand
double-nucleotide
HPS
mumet158 allele, not a more stable mRNA sectants (HPS
Lsmdrives
and HPS
dm, respectiveondary
structure,
the reduced
enzymatic
ly) were
transiently
transfected
to PC-12
activity
observed
for the
APS haplotype
(fig.
cells.
Asdifference
predictedisby
the mRNA
secondS4).
This
moderate
relative
to the
differencethe
coded
by
mRNA
structure–dependent
ary-structure
folding analyses,
HPS
LPS
HPS haplotypes.
were
Lsmand
exhibited
increased These
COMTresults
enzymatverified by an alternate approach of modifying
ic activity and protein levels equivalent to
mRNA secondary structure (fig. S5).
those of the LPS haplotype, whereas the
HPS dm exhibited reduced enzymatic
activity and protein levels equivalent to
those of the original HPS haplotype (Fig.
2, B and C; fig. S3, B and C). These data
rule out the involvement of RNA sequence
recognition motifs or codon usage in the
regulation of translation. In contrast to
the HPS haplotype, protein levels did not
parallel COMT enzymatic activity for the
APS haplotype and site-directed mutagenesis confirmed that the met158 allele,
not a more stable mRNA secondary structure, drives the reduced enzymatic activity observed for the APS haplotype (fig.
S4). This difference is moderate relative
to the mRNA structure–dependent difference coded by LPS and HPS haplotypes.
These results were verified by an alternate
approach of modifying mRNA secondary
structure (fig. S5).
Our data have very broad evolutionary
and medical implications for the analysis of
variants common in the human population.
The fact that alterations in mRNA secondary structure resulting from synonymous
changes have such a pronounced effect on
the level of protein expression emphasizes
the critical role of synonymous nucleotide
positions in maintaining mRNA secondary structure and suggests that the mRNA
secondary structure, rather than indepen-
162006
22 DECEMBER
VOL 314
SCIENCE
The fact that alterations in mRNA secondary
structure resulting from synonymous changes
have such a pronounced effect on the level of
protein expression emphasizes the critical role
of synonymous nucleotide positions in maindent nucleotides
in the synonymous
taining
mRNA secondary
structure andposisugtions, that
should
gests
theundergo
mRNA substantial
secondary selective
structure,
rather
than
independent
nucleotides
the
pressure
(22).
Furthermore,
our data in
stress
synonymous
positions,
should undergo
the importance
of synonymous
SNPssubas
stantial
selective
pressure
(22).inFurthermore,
potential
functional
variants
the area of
our data stress the importance of synonymous
humanas medical
SNPs
potential genetics.
functionalAlthough
variants innonthe
synonymous
are genetics.
believed Although
to have
area
of humanSNPs
medical
the strongest impact
nonsynonymous
SNPs on
arevariation
believed in
to gene
have
the
strongest
impact
on variation
in gene
function,
our data
clearly
demonstrate
that
function,
ourvariants
data clearly
demonstrate
that
haplotypic
of common
synonyhaplotypic variants of common synonymous
mous SNPs can have stronger effects on
SNPs can have stronger effects on gene funcgene than
function
than nonsynonymous
variation
nonsynonymous
variations
and
tionsan
and
play an important
role inonset
disease
play
important
role in disease
and
onset and progression.
progression.
References and Notes
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4. L. Diatchenko et al., Hum. Mol. Genet. 14, 135
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5. J. J. Marbach, M. Levitt, J. Dent. Res. 55, 711
(1976).
6. T. T. Rakvag et al., Pain 116, 73 (2005).
7. J. K. Zubieta et al., Science 299, 1240 (2003).
8. G. Winterer, D. Goldman, Brain Res. Brain Res. Rev. 43,
134 (2003).
9. B. Funke et al., Behav. Brain Funct. 1, 19 (2005).
10. G. Oroszi, D. Goldman, Pharmacogenomics 5, 1037
(2004).
11. J. Duan et al., Hum. Mol. Genet. 12, 205
(2003).
12. L. X. Shen, J. P. Basilion, V. P. Stanton Jr., Proc. Natl.
Acad. Sci. U.S.A. 96, 7871 (1999).
13. I. Puga et al., Endocrinology 146, 2210 (2005).
14. K. Mita, S. Ichimura, M. Zama, T. C. James, J. Mol. Biol.
203, 917 (1988).
15. T. D. Schmittgen, K. D. Danenberg, T. Horikoshi,
H. J. Lenz, P. V. Danenberg, J. Biol. Chem. 269, 16269
(1994).
16. A. Shalev et al., Endocrinology 143, 2541
(2002).
17. Materials and methods are available as supporting
material on Science Online.
18. D. H. Mathews, J. Sabina, M. Zuker, D. H. Turner, J. Mol.
Biol. 288, 911 (1999).
19. M. Zuker, Nucleic Acids Res. 31, 3406 (2003).
20. A. Y. Ogurtsov, S. A. Shabalina, A. S. Kondrashov,
M. A. Roytberg, Bioinformatics 22, 1317 (2006).
21. Sequence accession numbers: S-COMT clones BG290167,
CA489448, and BF037202 represent LPS, APS, and HPS
haplotypes, respectively. Clones corresponding to all
three haplotypes and including the transcriptional start
site were constructed by use of the unique restriction
enzyme Bsp MI. The gene coding region containing all
three SNPs was cut and inserted into the plasmids
through use of the S-COMT (BG290167) and MB-COMT
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onal
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onal
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esponse
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the NIH, National
for Biotechnology
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for
technical
support
and
W.
R.
Jacobs
at
Albert
Supporting
Online
Material
SOM Text
the NIH, National Center for Biotechnology REPORTS
Einstein
College
of
Medicine
for
providing
M.
tuber13 June
www.sciencemag.org/cgi/content/full/314/5807/1930/DC1
REPORTS
Figs.
S1 to200
S6
Information.
culosis H37Rv used in the study.
10.1126/scie
Materials
References
SOM Textand Methods
Supporting
Online
Material
SOM
Text
Figs.
to S6 2001; accepted 7 February 2002
10 S1
December
REPORTS
13 June 2006
www.sciencemag.org/cgi/content/full/314/5807/1930/DC1
Figs.
S1 to S6
References
10.1126/scien
Materials
and Methods
References
motes furt
SOM
13 Text
June 2006; accepted 16 November 2006
Figs.
to 2006;
S6
generation
13 S1
June
accepted 16 November 2006
10.1126/science.1131262
References
10.1126/science.1131262
ground in
Lineages
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Berkeley,
CA
USA.
Environmental
taining
PTCs
by
the94720,
NMD
pathway
effec[PTC(22)-PGK1],
and
nonstop-PGK1
population
members
ofA second
natural
consortia,
even
inthat
low
that
is related
to transcripts
ARMAN-1
(ARMAN-2)
was
independent
genomic
sequencing
approaches
assayed
PGK1
in
Saccharorors
in
mRNA
biogenesis.
The
decay
of
to
this
gene.
13.2-kb
genome
fragment
are
significantly
divergent
relative
to
gene
enes
(1–3).
reconstructed
from
117
Mb
of
community
geare
relatively
insensitive
to
organisms
that
sitology,
University
of
Queensland,
Brisbane
4072,
likely that low-abundance microorganisms dis- A drift (fig
Policy, and Management, University of California,
25, of Science,
Immunol.
es,
University
tively
prevents
expression
ofby
deleterious
was
created
by
removing
the
bona
fidegereconstructed
from
117
Mb
of
community
are
insensitive
tofor
organisms
complexity
systems
such
as
acid
mine
drainage
2fragments
Australia.
transcripts
containing
PTCs
the NMD
myces
cerevisiae
derived
from
the
followrRNA
gene
from
an
organism
a 16S
databases,
and
most
cultivationBerkeley,
CA
94720,
USA. 3Centre
Microscopy
and
nomic
sequence
derived
from
a biofilm
from
the site,Compa
occurrelatively
at in
low
abundance.
Consequently,
itthat
is encoding
es,
University of sequences
results
tantly
related
to known
species
will
be
undetected
Environmental
2
truncated
proteins.
In
prokaryotes,
protein
termination
codon
and all
termi(AMD)
(10).
nomic
sequence
derived
from
a biofilm
from
the fragments
occur
at
low
abundance.
Consequently,
it
is
Microanalysis
and
Department
of
Microbiology
and
Para42
(1996).
Environmental
is
related
to (3):
ARMAN-1
(ARMAN-2)
was
genomic
approaches
*Present
Universitysequencing
of California,
San Francisco,
A drift
(fig.
S1).
Within
the in-frame
data
sets from
each
likely address:
thateffectively
low-abundance
microorganisms
dis- that
ty of California, independent
pathway
prevents
expression
ing
constructs
wild-type
PGK1
(WTpopulation
members
of
natural
consortia,
even
in
low
Brenner,
B.
R.
products
encoded
by
transcripts
lacking
terUTR
(untranslated
nation
codons
in
the
3�
sitology,
University
of
Queensland,
Brisbane
4072,
A
drift
(fig.
S1).
Within
the
data
sets
from
each
likely
that
low-abundance
microorganisms
disinsertions
An
important
way
in
which
microorganisms
ty
of
California,
CAtantly
94143,
USA. toinsensitive
Microscopy and are
from
Mb
ofthat
community
gerelatively
to
organisms
that reconstructed
site, results
to
date117
indicate
eachharborARMAN
related
known species
will be undetected
Compa
complexity
systems
such
as of
acid
mine
drainage
of
deleterious
truncated
proteins.
InGenome
proPGK1),
afrom
nonsense
form
PGK1
University
of
Microscopy
and Australia.
mination
codons
are marked
foreven
degradaregion)
thecycles
WT-PGK1
transcript.
†Present
address:
Department
of
Energy
Joint
logy
and Paraaffect
geochemical
is by
accelerating
the in both o
site, results
date
indicate
each
ARMAN
tantly
tonatural
known
species
will
be
undetected
sequence
derived
from
athat
biofilm
from
the
atrelated
low
Consequently,
is nomic
population
istonear-clonal.
members
of abundance.
consortia,
init low
994).
nvironmental
fragments
(AMD)
(10).
logy and 4072,
Para- occur
Institute,
Walnut
Creek,
CA
94598,
USA.
karyotes,
protein
products
encoded
by
ing
a
PTC
at
codon
22
[PTC(22)-PGK1],
risbane
*Present
address:
University
of
California,
San
tion
by
thelow-abundance
addition
of
a COOH-terminal
transcripts
were
asand
labile
as
population
is near-clonal.
members
ofsystems
naturalsuch
consortia,
evenFrancisco,
indislow Nonstop-PGK1
pyrophosp
dissolution
of
minerals.
For
example,
microner,
Immunity
drift
(fig. S1).
Within
data
sets
from
each
that
microorganisms
of California,
Comparison
of the
ARMAN-1
-2
DNA
complexity
as
acidSanmine
risbane
4072, likely
insertions,
An
important
way
in the
which
microorganisms
‡Present
address:
University
of California,
Diego,drainage
La
Jolla, A
CA
94143,
USA.lacking
tag
encoded
by
tmRNA
(1,will
2).be
Thus,
both
their
nonsense-containing
counterparts
transcripts
termination
codons
are
and
nonstop-PGK1
that
was
created
by
organisms
can
derive
metabolic
energy
byDNA
ox- a gene sh
Comparison
ofindicate
the
ARMAN-1
and
-2
complexity
systems
such
as
acid
mine
drainage
croscopy and
site,
results
to
date
that
each
ARMAN
tantly
related
to
known
species
undetected
fragments
revealed
some
gene
rearrangements,
(AMD)
(10).
CA
92093,
USA.
†Present
address:
Department
of
Energy
Joint
Genome
in
both o
affect
geochemical
cycles
is
by
accelerating
the
San
Engl.
J. ParaMed.
the
presence
and CAcontext
of
translational
(Fig.
1). iron
At
least
three
trans-acting
factors
gy
andFrancisco,
fragments
revealed
some
gene
rearrangements,
(AMD)
(10).
idizing
released
by
the 1).dissolution
of Comparat
§
marked
for
byUSA.
the
addition
of population
removing
bona
fide
termination
codon
To
whom
correspondence
should
be
addressed.
Institute,
Creek,way
94598,
isthe
near-clonal.
of degradation
natural
consortia,
even
in E-mail:
low
San Francisco, members
insertions,
and
deletions
(Fig.
Genes
present
AnWalnut
important
in which
microorganisms
pyrophosp
dissolution
of
minerals.
For
example,
microbane 4072,
termination
can regulate
geneSan
expression.
(Upf1p,
Upf2p,
and
Upf3p)
essential
for
quences i
pyrite
). The
ferric
ironare
by-product
proinsertions,
deletions
(Fig.
1).
Genes
present
Anaddress:
important
way
‡Present
University
of in
California,
Diego,
La Jolla,
2and
995).
[email protected]
COOH-terminal
tag
encoded
by
tmRNA
and
all(FeS
in-frame
termination
codons
in
of
the
ARMAN-1
and
-2
DNA
systems
such
aswhich
acid
mine
drainage
Joint Genome complexity
inComparison
both
organisms
encode
putative
inorganic
affect
geochemical
cycles
is
by microorganisms
accelerating
the organisms
a gene sho
can
derive
metabolic
energy
by
oxInaffect
order
to determine
whether
the presencethe fragments
(4,
5).putative
Remarkably,
NMD
in S.
cerevisiae
CA
92093,
USA.
JointSpringer
Genome (AMD)
nner,
in
both
organisms
encode
inorganic
geochemical
cycles
is byexample,
accelerating
revealed
some
gene
rearrangements,
(10).
pyrophosphatases,
a
transcription
regulator,
and
dissolution
of
minerals.
For
microthe
3′
(1,
2).
Thus,
both
the
presence
and
context
UTR
(untranslated
region)
from
the
Comparati
idizing
iron
released
by
the
dissolution
of
Francisco,
Todissolution
whom
correspondence
shouldFor
be addressed.
E-mail:
translational
termination
influences
nonstop
transcripts
not
stabilized
inand
nanDiego,
La Jolla, §of
pyrophosphatases,
a were
transcription
regulator,
of derive
minerals.
example,
microand
deletions
(Fig.
1).
Genes
present
An
important
way
in metabolic
which microorganisms
a gene
shown
to be
an
arsenate
reductase
(13). quences in
organisms
can
energy
by
ox- insertions,
ARMAN-1
S. Jolla,
H.
pyrite
(FeS
). The
ferric
iron
by-product
[email protected]
nnegawa,
Diego, La
of
translational
termination
regulate
WT-PGK1
Nonstop-PGK1
tran2transcript.
mRNA
stability,
we metabolic
assayed can
PGK1
transtrains
lacking
Upf1p,
distinguishing
the
a
gene
shown
to
be
an
arsenate
reductase
organisms
can
derive
energy
by
oxoint
Genome
in
both
organisms
encode
putative
inorganic
affect
geochemical
cycles
is
by
accelerating
the
seidizing iron released by the dissolution of Comparative analysis of these genes with(13).
ressed. E-mail:
scripts
were
as
labile
their(Fig.
nonsensepathway
of decay
fromofas
NMD
1).with pyrophosphatase
Comparative
analysis
these
genes
idizing
iron
released
byiron
the
dissolution
of pyrophosphatases,
838
(1995).
ressed.
E-mail: dissolution
apublic
transcription
regulator,
andseof2).minerals.
example,
microquences in the
databases
consistently
pyrite (FeS
The ferricFor
by-product
pro1
2
49
(1992).
1
Institute
for Genetic
Medicine,
Department
of
BioThe
turnover
of
normal
mRNAs
requires
iego,
La Jolla,
ARMAN-1
containing
counterparts
(Fig.
1).
At
least
quences
in
the
public
databases
consistently
pyrite
(FeS
).
The
ferric
iron
by-product
pro2 derive metabolic energy by oxa gene shown to be an arsenate reductase (13).
organisms
1217 (1997).
physics andcan
Biophysical
Chemistry, Johns Hopkins
deadenylation
followed by (Upf1p,
Dcp1p-mediatthree trans-acting
L. Hendricks,
analysisfactors
of these genes Upf2p,
with seidizing
released
by Baltimore,
the dissolution
of Comparative
pyrophosphatase i
Universityiron
School
of Medicine,
MD 21205,
ssed.
E-mail:
ARMAN-1
ed decapping and degradation by
the major
3
(1997).
90%
31%
69%
85%
USA.
University
of
Arizona,
Department
of
Molecular
1 for NMD
quences
in the
public
databases
consistently
pyrite (FeS2). The ferric iron by-product proand
Upf3p)
are
essential
in
S.
ARMAN-1
wstead, T. J.
5�-to-3� exonuclease
Xrn1p (6, 7 ). NMD is
and Cellular Biology, Tucson, AZ 85721, USA.pyrophosphatase
insert
cerevisiae
(4,
5).
Remarkably,
nonstop
00).
4
pyrophosphatase
Howard Hughes Medical Institute.
distinguished
insertin that these events occur
1
(1999).
4,226
90%
31%in
69% 85%
transcripts
were
not
stabilized
1
1 correspondence should
without
prior
deadenylation
(8,
9).strains
Non4,226
*To whom
be addressed.ARMAN-1
E143 (2000).
arsC
transposase GTP cyclohydrolases ribosomal proteins mercuric reductase
mail: [email protected]
cience Online
stop-PGK1
transcripts
showed the
rapid
decay
lacking Upf1p,
distinguishing
pathway
pyrophosphatase
insert
O
O
E
31%
1
69% 85%
90%
97%
97%
90%
1
31%
69% 85%ARMAN-2
4,226
transposase
GTP cyclohydrolases
ribosomal proteins mercuric reductase
VOL
295 SCIENCE
www.sciencemag.org
22 MARCH 2002
Fig. 1. Comparison of syntenous31%
genomic regions
of ARMAN-1
97%the
90% [from
69% 85%
ydrolases ribosomal proteins mercuric reductase
arsC
ARMAN-2 16S rRNA
arsC
17
percentage13,236
similarity for the 16S
13,236
of decay from NMD (Fig. 1).
The turnover of normal mRNAs requires deadenylation followed by Dcp1pmediated decapping and degradation by
the major 5′-to-3′ exonuclease Xrn1p (6,
7). NMD is distinguished in that these
events occur without prior deadenylation
(8, 9). Nonstop-PGK1 transcripts showed
rapid decay in strains lacking Xrn1p or
Dcp1p activity (Fig. 1), providing further evidence that they are not subject to
NMD. This result was surprising since
both of these factors are also required
for the turnover of normal mRNAs after
deadenylation by the major deadenylase
Ccr4p (10). Nonstop decay was also un-
Fig. 1. Nonstop-PGK1 transcripts are rapidly
degraded by a novel mechanism. Half-lives
(t1/2) of WT-PGK1, PTC(22)-PGK1, nonstopPGK1, and Ter-poly(A)-PGK1 transcripts
are shown (27). Half-lives were performed
in a wild-type yeast strain ( WT ), strains
deleted for Upf1p (upf1Δ), Xrn1p (xrn1Δ), or
Ccr4p (ccr4Δ), a strain lacking activity of the
decapping enzyme Dcp1p (dcp1-2), and a WT
yeast strain treated for 1 hour with 100 μg/ml
of cycloheximide (+CHX ).
18
altered in a strain lacking Ccr4p (Fig. 1).
Therefore, degradation of nonstop-PGK1
transcripts requires none of the factors involved in the pathway for degradation of
wild-type or nonsense mRNAs.
Additional experiments were performed
to assess the role of translation in nonstop
decay. Treatment with cycloheximide
(CHX) or depletion of charged tRNAs in
yeast harboring the conditional cca1-1 allele (11) grown at the nonpermissive temperature substantially increased the stability of nonstop-PGK1 transcripts (Fig. 1)
(12). It has been shown that translation
into the 3′ UTR of selected transcripts can
displace bound trans-factors that are posi-
tive determinants of message stability (13,
14). To determine whether this mechanism
is relevant to nonstop decay, we examined
into the 3′ UTR of selected transcripts can
substantial (threefold) stabilization (Fig. 1).
Unlike nonstop-PGK1 transcripts, the halflife of Ter-poly(A)-PGK1 was increased in
the absence of Xrn1p (Fig. 1), indicating
that
transcript isofdegraded
by the pathtivethis
determinants
message stability
(13,
way
normal mRNA
by the
14).for
To determine
whetherand
thisnot
mechanism
nonstop pathway. These data suggest that
is relevant to nonstop decay, we examined
the instability of nonstop-PGK1 transcripts
the performance
of transcript
Ter-poly(A)cannot
fully be explained
by ribosomal
disPGK1
which
contains
a
termination
UTR.
placement of factors bound to the 3� codon
inserted
oneancodon
upstream
the the
site 5�
of
There is
emerging
viewofthat
to form
a
and
3� ends of mRNAs
polyadenylate
[poly(A)]interact
addition
(15) in
closed
loop nonstop-PGK1.
and that this conformation
transcript
The addition is
of
required
for efficient
initiation
a termination
codon translation
at the 3′ end
of the
and normal mRNA stability. Participants in
3′ UTR of a nonstop transcript resulted in
the interaction include components of the
substantial
(threefold)
stabilization
(Fig.
translation
initiation
complex
eIF4F as well
1).
Unlike
nonstop-PGK1
transcripts,
the
as Pab1p. The absence of a termination
half-life
of Ter-poly(A)-PGK1
was
incodon
in nonstop-PGK1
is predicted to
allow
thecreased
ribosome
to continue
through
in the
absence translating
of Xrn1p (Fig.
1),
tail, potentially
retheindicating
3� UTR and
that poly(A)
this transcript
is degraded
sulting
displacement
of Pab1p,
disruption
by theinpathway
for normal
mRNA
and not
of normal mRNP structure, and consequently
by the nonstop pathway. These data suggest that the instability of nonstop-PGK1
transcripts cannot fully be explained by
ribosomal displacement of factors bound
to the 3′ UTR.
There is an emerging view that the
5′and 3′ ends of mRNAs interact to form
a closed loop and that this conformation is
required for efficient translation initiation
and normal mRNA stability. Participants
in the interaction include components of
the translation initiation complex eIF4F as
well as Pab1p. The absence of a termination codon in nonstop-PGK1 is predicted
to allow the ribosome to continue translating through the 3′ UTR and poly(A)
tail, potentially resulting in displacement
of Pab1p, disruption of normal mRNP
structure, and consequently accelerated
degradation of the transcript. However,
while mRNAs in pab1Δ strains do undergo accelerated decay, the rapid turnover is
a result of premature decapping and can
be suppressed by mutations in XRN1 (16),
allowing distinction from nonstop decay.
Lability of nonstop transcripts might also
e rapidly
Half-lives
be a consequence of ribosomal stalling at
nonstopthe 3′ end of the transcript. It is tempting
ipts are
to
speculate that 3′-to-5′ exonucleolytic
ed in a
leted for
activity underlies the decapping- and 5′r Ccr4p
to-3′ exonuclease–independent, translae decaption-dependent
accelerated decay of nonWT yeast
/ml of cycloheximide
(�CHX ). An appealing candidate
stop transcripts.
lowing distinction from nonstop decay. Lability of nonstop transcripts might also be a
consequence of ribosomal stalling at the 3�
end of the transcript. It is tempting to specuexonucleolytic
activity
late
3�-to-5�a collection
is thethat
exosome,
of proteins
exounderlies
the exoribonuclease
decapping- and 5�-to-3�
with 3′-to-5′
activity that
nuclease–independent, translation-dependent
functions in the processing of 5.8S RNA,
accelerated decay of nonstop transcripts. An
rRNA, small
nucleolar
(snoRNA),
appealing
candidate
is the RNA
exosome,
a collecsmall
nuclear
RNA
(snRNA),
and other
tion of proteins with 3�-to-5� exoribonuclease
transcripts.
presented
van Hoof etof
activity
that Data
functions
in thein processing
5.8S
RNA,
rRNA,
small nucleolar RNA
al. (17)
validate
this prediction.
Downloaded from www.sciencemag.org on June 29, 2007
e major
p decay
g Ccr4p
nonstopthe facdegradaNAs.
rformed
nonstop
eximide
NAs in
a1-1 alve temstabili(Fig. 1)
ion into
can dispositive
13, 14 ).
nism is
ined the
poly(A)n codon
Fig. 2. Nonstop decay is conserved in mammalian cells. (A) Steady-state abundance of �-glucuronidase transcripts derived from wild type–
( WT ), nonsense-, nonstop-, and Ter-poly(A)�gluc minigene constructs after transient
transfection into HeLa cells (28). Zeocin (zeo)
transcripts served as a control for loading and
transfection efficiency. For each construct, the
�gluc/Zeo transcript ratio is expressed relative
to that observed for WT-�gluc. (B) Steadystate abundance of WT-�gluc, nonstop-�gluc,
and nonsense-�gluc transcripts were determined in the nuclear (N) and cytoplasmic (C)
fractions of transfected cells (28). The ratio of
normalized nonstop- or nonsense-�gluc transcript levels to WT-�gluc transcript levels are
shown for each cellular compartment. Reported
results are the average of three independent
experiments.
www.sciencemag.org SCIENCE VOL 295 22 MARCH 2002
19 2259
In order to determine whether nonstop
decay functions in mammals, we assessed
the performance of transcripts derived
from β-glucuronidase (βgluc) minigene
constructs containing exons 1, 10, 11, and
12 separated by introns derived from the
endogenous gene (18). The abundance of
transcripts derived from a nonstop-βgluc
version of this minigene was significantly
reduced relative to WT-βgluc, suggesting
that a termination codon is also essential
for normal mRNA stability in mammalian cells (Fig. 2A). Addition of a stop
codon one codon upstream from the site
of poly(A) addition in nonstop-βgluc
[Ter-poly(A)-βgluc (15)] increased the
abundance of this transcript to near–wildtype levels (Fig. 2A). Thus, all functional
characteristics of nonstop transcripts in
yeast appear to be relevant to mammalian
systems.
Both the abundance and stability of
most nonsense transcripts, including nonsense-βgluc (Fig. 2B), are reduced in the
nuclear fraction of mammalian cells (19,
20). This has been interpreted to suggest
that translation and NMD initiate while
the mRNA is still associated with (if
not within) the nuclear compartment. If
translation is initiated on nucleus-associated transcripts, so might nonstop decay.
Subcellular fractionation studies localized
mammalian nonstop decay to the cytoplasmic compartment, providing further
distinction from NMD (Fig. 2B).
The conservation of nonstop decay
in yeast and mammals suggests that the
pathway serves an important biologic
role. There are many potential physiologic
sources of nonstop transcripts that warrant
consideration. Mutations in bona fide termination codons would not routinely initiate nonstop decay due to the frequent occurrence of in-frame termination codons in
the 3′ UTR and could not plausibly provide
the evolutionary pressure for maintenance
of nonstop decay. In contrast, alternative
use of 3′-end processing signals embedded
in coding sequence has been documented
20
in many genes including CBP1 in yeast
and the growth hormone receptor (GHR)
gene in fowl (21–23). Moreover, a computer search of the human mRNA and S.
cerevisiae open reading frame (ORF) databases revealed many additional genes that
contain a strict consensus sequence for 3′
end cleavage and polyadenylation within
their coding region (Fig. 3A). Utilization
of these premature signals would direct
formation of truncated transcripts that
might be substrates for the nonstop decay
pathway. Indeed, analysis of 3425 random
yeast cDNA clones sequenced from the 3′
end (i.e., 3′ ESTs) revealed that 40 showed
apparent premature polyadenylation within the coding region (24).
The truncated GHR transcript in fowl
is apparently translated, as evidenced by
its association with polysomes (22). As
predicted for a substrate for nonstop decay, the ratio of truncated–to–full-length
GHR transcripts was dramatically increased after treatment of cultured chicken hepatocellular carcinoma cells with the
translational inhibitor emetine (Fig. 3B).
Other truncated transcripts, both bigger
and smaller than the predicted 0.7-kb nonstop transcript, did not show a dramatic
increase in steady-state abundance upon
translational arrest suggesting that they
are derived from other mRNA processing
events, perhaps alternative splicing (Fig.
3B) (12). To directly test whether the nonstop mRNA pathway degrades prematurely polyadenylated mRNAs, we analyzed
CBP1 transcripts in yeast. The CBP1 gene
produces a 2.2-kb full-length mRNA and
a 1.2-kb species with premature 3′ end
processing and polyadenylation within the
coding region (25). As expected from the
observation that nonstop decay requires
the exosome in yeast (17), deletion of the
gene encoding the exosome component
Ski7p stabilized the prematurely polyadenylated mRNA but had no effect on the
stability of the full-length mRNA (Fig.
3C). These data indicate that physiologic
transcripts arising from premature poly-
Fig. 3. Many physiologic transcripts are
substrates for nonstop decay. (A) 239
human mRNAs contain a polyadenylation
signal consisting of either of the two most
common words for the positioning, cleavage,
and downstream elements (29), separated by
optimal distances (30), where the cleavage
site occurs between the start and stop
codons as determined from the cDNA coding
sequence (CDS). Similarly 52 S. cerevisiae ORFs
contained the yeast polyadenylation signal
consisting of optimal upstream, positioning,
and cleavage signals followed by any of nine
common “U-rich” signals (24) with optimal
spacing of the elements. Computer search
was performed using the human mRNA
database and the S. cerevisiae ORF database
and an analysis program written in PERL that
is available upon request. (B) Abundance of
nonstop and full-length cGHR transcripts in
chicken hepatocellular carcinoma cells (CRL2117 purchased from the American Type
Culture Collection, Manassas, VA) treated
with 100 μg/ml of emetine for 6 hours. Northern analysis was performed as described (22). The
relative ratio of each transcript in untreated and treated cells is reported. (C) Half-lives (t1/2) of
the full-length and nonstop CBP1 mRNAs measured in wild-type–(WT) and SKI7-deleted (ski7Δ)
yeast strains carrying plasmid pG::-26 (25, 31). (D) Half-lives of WT-PGK1 and Ter-poly(A)-PGK1
transcripts in wild-type–(WT ) and SKI7-deleted (ski7Δ) yeast strains treated with the indicated
dose of paromomycin (mg/ml) (PM) for 20 hours. For cycloheximide (CHX ) experiments, yeast
were treated identically except 100 μg/ml of CHX was added during the last hour before half-life
determination. Half-lives were determined as described (27).
21
adenylation are subject to nonstop mRNA
decay. The cytoplasmic localization of
ski7p is consistent with our observation
that nonstop decay occurs within the cytoplasm (Fig. 2B).
Any event that diminishes translational
fidelity and promotes readthrough of termination codons could plausibly result in
the generation of substrates for nonstop
decay. In view of recent attempts to treat
genetic disorders resulting from PTCs
with long-term and high-dose aminoglycoside regimens, this may achieve medical
significance. As a proof-of-concept experiment, we examined the performance of
transcripts with one [Ter-poly(A)-PGK1]
or multiple (WT-PGK1) in-frame termination codons (including those in the 3′
prematurely polyadenylated mRNA but had
UTR)
in yeast
after
with
no
effect
on thestrains
stability
of treatment
the full-length
paromomycin,
which
ribosomal
mRNA
(Fig. 3C).
Theseinduces
data indicate
that
readthrough.transcripts
Remarkably,
bothfrom
transcripts
physiologic
arising
premature
polyadenylation
are subject
to nonstop
showed
a dose-dependent
decrease
in stamRNA
decay.
Thebecytoplasmic
bility that
could
reversed bylocalization
inhibiting
of ski7p is consistent with our observation
translational elongation with CHX or prethat nonstop decay occurs within the cytovented(Fig.
by deletion
of the gene encoding
plasm
2B).
Ski7p
data suggest
that
Any(Fig.
event3D).
that These
diminishes
translational
nonstopand
decay
can limit
the efficiency
of
fidelity
promotes
readthrough
of termination
codons
could plausibly
in the
therapeutic
strategies
aimed atresult
enhancing
generation
of substratesand
for nonstop
decay.
nonsense suppression
might contribIn view of recent attempts to treat genetic
ute to the toxicity associated with aminodisorders resulting from PTCs with longglycoside
therapy. aminoglycoside regiterm
and high-dose
Thethis
degradation
of nonstop
transcripts
mens,
may achieve
medical
significance.
a proof-of-concept
may beAs
regulated.
The relativeexperiment,
expression
we
examined
the performance
of transcripts
level
of truncated
GHR transcripts
comwith
[Ter-poly(A)-PGK1]
multiple
paredone
to full-length
transcriptsor
varies
in a
(WT-PGK1) in-frame termination codons
tissue-, gender-, and developmental stage(including those in the 3� UTR) in yeast
specificafter
manner
(22) and
theparomomycin,
relative abunstrains
treatment
with
dance of
truncated
CBP1 readthrough.
transcripts varies
which
induces
ribosomal
Remarkably,
both
transcripts
showed
a dosewith growth
condition
(21).
Data presentdependent
decrease
in stabilitythat
thatregulacould
ed here warrant
the hypothesis
be
reversed
by inhibiting
translational
tion
may occur
at the level
of nonstop elontrangation with CHX or prevented by deletion
script stability rather than production. The
of the gene encoding Ski7p (Fig. 3D).
conservation
of the that
GHR
codingdecay
sequence
These
data suggest
nonstop
can
3′- end
throughout
avian
limit
theprocessing
efficiency signal
of therapeutic
strategies
aimed
at enhancing
nonsenseofsuppression
phylogeny
and conservation
premature
and
might contribute
to yeast
the toxicity
associpolyadenylation
of the
RNA14
tranated
therapy.
scriptwith
andaminoglycoside
its fruitfly homolog
su(f ) supThe degradation of nonstop transcripts may
port speculation that nonstop transcripts
be regulated. The relative expression level of
or derivedGHR
protein
products
serve essential
truncated
transcripts
compared
to fulllength transcripts varies in a tissue-, gender-,
and
22 developmental stage-specific manner (22)
and the relative abundance of truncated CBP1
developmental and/or homeostatic functions that are regulated by nonstop decay
(21, 26).
Many processes contribute to the precise control of gene expression including
transcriptional and translational control
mechanisms. In recent years, mRNA stability has emerged as a major determinant
of both the magnitude and fidelity of gene
expression. Perhaps the most striking and
comprehensively studied example is the
accelerated decay of transcripts harboring
PTCs by the NMD pathway. Nonstop decay now serves as an additional example
of the critical role that translation plays
in monitoring the fidelity of gene expression, the stability
R E PofOaberrant
R T S or atypical
transcripts, and hence the abundance of
gene expression, the stability of aberrant or
truncated
proteins. and hence the abundance
atypical
transcripts,
of truncated proteins.
References and Notes
1. A. W. Karzai, E. D. Roche, R. T. Sauer, Nature Struct.
Biol. 7, 449 (2000).
2. K. C. Keiler, P. R. Waller, R. T. Sauer, Science 271, 990
(1996).
3. The nonstop-PGK1 construct was generated from
WT-PGK1 [pRP469 (8)] by creating three point mutations, using site-directed mutagenesis (QuikChange Site-Directed Mutagenesis Kit, Stratagene)
which eliminated the bona fide termination codon
and all in-frame termination codons in the 3� UTR.
The Ter-poly(A)-PGK1 construct was created from
the nonstop-PGK1 construct, using site-directed mutagenesis (Quik-Change Site-Directed Mutagenesis
Kit, Stratagene). All changes were confirmed by sequencing. Primer sequences are available upon request. The PTC(22)-PGK1 construct is described in (8)
( pRP609).
4. P. Leeds, J. M. Wood, B. S. Lee, M. R. Culbertson, Mol.
Cell. Biol. 12, 2165 (1992).
5. Y. Cui, K. W. Hagan, S. Zhang, S. W. Peltz, Genes Dev.
9, 423 (1995).
6. D. Muhlrad, C. J. Decker, R. Parker, Genes Dev. 8, 855
(1994).
7. C. A. Beelman et al., Nature 382, 642 (1996).
8. D. Muhlrad, R. Parker, Nature 370, 578 (1994).
9. G. Caponigro, R. Parker, Microbiol. Rev. 60, 233
(1996).
10. M. Tucker et al., Cell 104, 377 (2001).
11. C. L. Wolfe, A. K. Hopper, N. C. Martin, J. Biol. Chem.
271, 4679 (1996).
12. P. A. Frischmeyer, H. C. Dietz, data not shown.
13. I. M. Weiss, S. A. Liebhaber, Mol. Cell. Biol. 14, 8123
(1994).
14. X. Wang, M. Kiledjian, I. M. Weiss, S. A. Liebhaber,
Mol. Cell. Biol. 15, 1769 (1995).
15. We performed 3� rapid amplification of cDNA ends
(RACE) using the Marathon cDNA Amplification Kit
(Clontech) and the Advantage 2 Polymerase Mix
(Clontech) according to the manufacturer’s instructions. Poly(A) RNA isolated from cells (Oligotex
mRNA midi kit, Qiagen) transiently transfected with
WT-�gluc or from a wild-type yeast strain carrying a
plasmid expressing WT-PGK1 was used as template.
A single RACE product was generated, cloned ( TOPO
TA 2.1 kit, Invitrogen), and sequenced.
16. G. Caponigro, R. Parker, Genes Dev. 9, 2421 (1995).
21. K. A. Spa
4676 (19
22. E. R. Ol
Endocrin
23. C. Hilge
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Nucleic
25. K. A. Spa
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26. A. Audib
U.S.A. 9
27. All PGK1
the GAL1
were gro
media–u
scription
SC-ura m
off ) to a
removed
10 �g of
od (4) w
dehyde g
Screen P
nucleotid
cally det
transcrip
the perce
log plot.
for the c
formed a
1 hour] e
28. HeLa ce
bovine s
confluen
RPMI 16
dithiothr
and cells
Electrop
a capaci
kit, Qiag
tion, wa
gel, tran
with a po
prising e
had bee
(Random
heim). T
with boi
labeled
quantita
subcellu
HeLa cel
Downloaded
www.sciencemag.org
on
June
29, 2007
Downloaded
fromfrom
www.sciencemag.org
on June
29, 2007
Downloaded
from www.s
(Quik-Change
Site-Directed
Mutagenesis
The
Ter-poly(A)-PGK1
was of
created
amplification
cDNA from
ends
15. tagenesis
We performed
3� rapidconstruct
removed
at theGlucose
indicated
gel, transferred
to
a nylon
membrane,
and
hybridized
ity
associSC-ura
media.
wastime
thenpoints.
addedApproximately
(transcription
at genetic
Kit,
Stratagene).
changes cDNA
were
bymuseside
regithe
nonstop-PGK1
construct,
using confirmed
site-directed
(RACE)
using theAllMarathon
Amplification
Kit
�g
isolated
with
the
hot phenol
10
with
aofpolymerase
chain reaction
(PCR)
productmethcomoff
) to
a total
final RNA
concentration
of 2%
and
aliquots
were
y. longwith
quencing.
Primer
sequences
are
available
upon
retagenesis
Site-Directed
Mutagenesis
(Clontech)(Quik-Change
and the Advantage
2 Polymerase
Mix
on of
a points.
1.2%
agarose
formalod
(4) was
l signifigene
that
prising
exons
through time
12
the �-gluc
removed
atelectrophoresed
the10indicated
Approximately
quest.
The
PTC(22)-PGK1
construct
is
described
in
(8)
scripts
may
Kit,
Stratagene).
All
changes
were
confirmed
by
seside
regi(Clontech) according to the manufacturer’s instrucdehyde
transferred
tolabeled
awith
nylon
membrane
(Genehad�gbeen
radioactively
by
nick
translation
ofgel,
total
RNA isolated
the
hot
phenol
meth10
periment,
(
pRP609).
quencing.
Primer
sequences
are
available
upon
retions.
Poly(A)
RNA
isolated
from
cells
(Oligotex
Screen
Plus,
NEN),DNA
and Labeling
hybridized
with
a 23 bpformaloligoon signifilevel of
(Random
Primed
Boehringer
Mannod
(4) was
electrophoresed
on a Kit,
1.2%
agarose
al
ranscripts
4. P.
Leeds,
J. PTC(22)-PGK1
M.kit,
Wood,
B. S.transiently
Lee, M. R.isCulbertson,
Mol.
quest.
The
construct
described in
(8)
mRNA
midi
Qiagen)
transfected
with
nucleotide
probe
that stripped
specifiheim). gel,
Theend-labeled
membraneradioactive
subsequently
dehyde
transferred
towas
a nylon
membrane
(Geneed
to fullperiment,
2165a(1992).
Cell.
Biol. 12,
(WT-�gluc
pRP609).
multiple
or from
wild-type yeast strain carrying a
UTR
of the
PGK1
cally
the
polyG
the 3�
withdetects
boiling
0.5%
SDS
and in
probed
with
Screen
Plus, NEN),
andtract
hybridized
with
a radioactively
23
bp oligo-, gender-,
5.
Cui,
K. expressing
S.B.Zhang,
W.R.used
Peltz,
Genes
Dev.
ranscripts
4. Y.
P.
Leeds,
J.W.M.Hagan,
Wood,WT-PGK1
S. Lee,S.M.
Culbertson,
Mol.
plasmid
was
as
template.
Half-lives
were determined
by plotting
transcripts
(8).
labeled zeocin
cDNA.radioactive
Northern
blot results
were
nucleotide
end-labeled
probe
that
specifin codons
9,
423
(1995).
12,
2165
(1992).
Cell.
Biol.
(22)
A single RACE product was generated, cloned ( TOPO
the
percent
mRNA
versus
time
a semiquantitated
using
anremaining
Instant
Imager
(Packard).
For
ranner
multiple
ofonthe
PGK1
cally
detectsofthe
polyG
tract
in the
3� UTR
in
yeast
8, Dev.
855
6.
D.
Muhlrad,
C.Hagan,
J. Decker,
R. Parker,
Genes
Dev.
5.
Y.
Cui,
K.
W.
S.
Zhang,
S.
W.
Peltz,
Genes
TA
2.1
kit,
Invitrogen),
and
sequenced.
log
plot.
All
half-lives
were
determined
at
25°C
except
subcellular
fractionation,
transiently
transfected
transcripts (8). Half-lives were determined by plotting
ated
CBP1
n codons
9,
(1995). R. Parker, Genes Dev. 9, 2421 (1995).
momycin,
( performed
at washed
30°C)
and
dcp1-2
[perfor
the
ccr4�
16. (1994).
G.423
Caponigro,
phosHeLa
cells
were
trypsinized,
intime
cold
1�
the
percent
of
mRNA
remaining
versus
on
a
semidition
(21).
Nature
382,
(1996).
7.
Beelman
al.,
) in yeast
855
6. C.
D.
Muhlrad,
J.A.
Decker,
R. Parker,
Genes
Dev.
formed
after
shiftsaline,
to the
nonpermissive
(37°C)
for
17.
A. A.
van
Hoof,C.P.et
Frischmeyer,
H. 642
C. Dietz,
R. 8,
Parker,
phate
and
pelleted bytemp
centrifuging
at
log
plot.buffered
All half-lives
were
determined
at
25°C
except
ough.
Re8. D.
Muhlrad,
Parker,
Nature 370, 578 (1994).
othesis
that
(1994).
1for
hour]
experiments.
295,R.2262
(2002).
Science
5 min
at 4°C. Cells
were then
resuspended
2040g
momycin,
(performed
at 30°C)
and dcp1-2
[perthe for
ccr4�
d a dose60,
233
9.
G.
Caponigro,
R.
Parker,
Microbiol.
Rev.
Nature
382,
642
(1996).
7.
C.
A.
Beelman
et
al.,
28.
HeLa
cells
[maintained
in
DMEM
with
10%
fetal
minigene
construct,
mouse
18.
To
create
the
WT-�gluc
,
10
mM
�l
of
140
mM
NaCl,
1.5
mM
MgCl
in
200
formed after shift to the nonpermissive temp (37°C)
for
of nonstop
2
ough.
Rehat could
370, 578for
(1994).
8. (1996).
D.
Muhlrad,
R. Parker,
Nature
to 90%
bovine
were NP-40,
grown to
genomic
DNA
was used
as template
amplifying
( pH (FBS)]
8.6), 0.5%
and�80
1 mM
DT T,
1Tris-HCl
hour] serum
experiments.
uction.
The 10.
d a dose104,
Tucker
et al.,portions
233
9. M.
G.
Caponigro,
R.Cell
Parker,
Microbiol.
Rev. 60, gene:
of
trypsinized,
resuspended
300 �l
�-glucuronidase
three
different
of 377
the (2001).
vortexed,
incubatedand
on
for 5 with
min.in
Nuclei
were
onal
elon28. confluency,
HeLa
cellsand
[maintained
in ice
DMEM
10%
fetal
L. Wolfe,
A. K. 2313
Hopper,
C. Martin,
J. Biol. Chem.
quence
3�- 11. C.
(1996).
RPMI
1640
10
glucose
and
0.1
mM
could
from
nucleotide
to N.
3030
encompassing
exon 1
45 sto
at90%
4°C
pelleted
bywithout
centrifuging
at mM
12,000g
�80
bovine
serum
(FBS)]FBS,
were
grown
tofor
yhat
deletion
271,
4679
(1996).
Cell
104,
377
(2001).
10.
M.
Tucker
et
al.,
�g)
was
added
dithiothreitol
(DT
T).
Plasmid
DNA
(10
and
part
of
intron
1
(including
the
splice
donor
seand subsequently
separated
from the aqueous
an phylog�l of
confluency,
trypsinized,
and resuspended
in 300(cytoonal
elonFig.
3D).
12.
A.
Frischmeyer,
H. C. Dietz,
notJ.shown.
and
cells
were
electroporated
(Bio-Rad,
II
11. P.
C.
L. Wolfe,
A. nucleotide
K. Hopper,
N. C.data
Martin,
Biol.
Chem.
quence),
from
11285
to 13555
encompassplasmic)
phase.
RPMI
1640
without
FBS, 10 mM
glucoseGene
and Pulser
0.1 mM
yolyadenyldeletion
14, 8123
13. I.271,
M. part
Weiss,
S. A. Liebhaber,
Mol.
Biol.
System)
atS.aC.voltage
of�g)
0.300
and
4679
ing
of (1996).
intron
9 (including
theCell.
splice
acceptor
se29. Electroporation
J. H. Graber, C.(DT
R.
Cantor,
Mohr,
F.
Smith,
Proc.
decay
can
waskV
added
dithiothreitol
T).
Plasmid
DNA
(10T.
ipt
and
its
(1994).
Fig.
3D).
�F.96,
Poly(A)
RNA
(Oligotex
midiII
aand
capacitance
of
500
12. P.
A. Frischmeyer,
Dietz,
data not
shown. the
quence
), exon 10, H.
andC.part
of intron
10 (including
14055
(1999).
Natl.
Acad.
Sci.
U.S.A.
cells
were
electroporated
(Bio-Rad,
Gene Pulser
strategies
Wang,
M.S.Kiledjian,
I. and
M.Mol.
Weiss,
S. Biol.
A. Liebhaber,
�g),
isolated
36
hours
transfecQiagen,
donor
sequence),
from
13556
to
14,15813
8123
13. X.
I.splice
M.
Weiss,
A. Liebhaber,
Cell.
30. kit,
F. Chen,
C. C.2 MacDonald,
Nucleic
Acids
Res.
ulation
that 14.
Electroporation
System)
atJ.aWilusz,
voltage
ofafter
0.300
kV and
decay
can
ppression
1769
(1995).
Mol.
Cell.
Biol. 15,
tion,
was
separated
on
a
1.6%
agarose
formaldehyde
which
included
the
remainder
of
intron
10,
exon
11,
(1994).
23,
2614
(1995).
a capacitance of 500 �F. Poly(A) RNA (Oligotex midi
nstrategies
products 15. We performed 3� rapid amplification of cDNA ends
transferred
to(25)
a isolated
nylon
membrane,
and
ty
associintron
11, M.
andKiledjian,
exon 12.I.All
fragments
were TA
14. X.
Wang,
M.three
Weiss,
S. A. Liebhaber,
31. gel,
Plasmid
pG-26
containing
the CBP1
gene un36 hours
afterhybridized
transfeckit,
Qiagen,
2 �g),
or homeo(RACE)
using
cDNA Amplification
Kit
with
awas
polymerase
reaction
(PCR) formaldehyde
product
cloned
( Topo
TA
2.1
kit, (1995).
Invitrogen),
sequenced, and
15,Marathon
1769
Mol.
Cell.
Biol.the
der the
control
ofchain
the
promoter
and the comLEU2
tion,
separated
on aGAL
1.6%
agarose
y.ppression
Advantage
2 Polymerase
Mix
gene
that
prising
exonsmarker
10tothrough
12
of the �-gluc
by
thenperformed
clonedand
into3�the
pZeoSV2
(Invitrogen).
single baserapid
amplification
ofA cDNA
ends
15. (Clontech)
We
selectable
was introduced
into
wild-type
gel,
transferred
a nylon
membrane,
and hybridized
ty nonstop
associcripts
may
(Clontech)
according
to the to
manufacturer’s
instruchad
been
radioactively
labeled
by
nick
translation
was
pair deletion
corresponding
the Amplification
gusmps allele
(RACE)
using
the
Marathon
cDNA
Kit
and
SKI7-deleted
strains.
Yeast
were
grown
in
mewith a polymerase chain reaction (PCR) product comy.n level of
tions.
Poly(A)
RNA
isolated
from
cells
(Oligotex
(Random
Primed
Labeling
Manngenerated
by
site-directed
mutagenesis
(Quik-Change
(Clontech)
and
the Advantage
2 Polymerase
Mix
dia lacking
leucine
and
containing
2% galactose
at
�-gluc
gene
that
prising
exons
10 DNA
through
12 ofKit,
theBoehringer
he precise
cripts
may
mRNA
midiaccording
kit,
Qiagen)
transfected
with
heim).
membrane
was
subsequently
stripped
Site-Directed
Mutagenesis
Kit,manufacturer’s
Stratagene)
of the
WT(Clontech)
to transiently
the
instrucCells
were
and
30°Cbeen
toThe
anradioactively
OD600 of 0.5.
had
labeled
by
nickpelleted
translation
ed to fullor
from
a
wild-type
yeast
strain
carrying
a
WT-�gluc
ding
tranwith
boiling
0.5%
SDS
andlacking
probed
radioactively
�gluc construct
to create
nonsense-�gluc.
Nonstoptions.
Poly(A) RNA
isolated
from cells (Oligotex
resuspended
in DNA
media
aBoehringer
carbon
source.
level of
(Random
Primed
Labeling
Kit, with
Mann-,n gender-,
plasmid
expressing
WT-PGK1
was used
as template.
labeled
zeocin
cDNA.
Northern
results
were
�gluc was
generated
fromtransiently
WT-�gluc
by two
rounds
of
mRNA
midi
kit, Qiagen)
transfected
with
GlucoseThe
wasmembrane
then
added
to asubsequently
finalblot
concentration
of
heim).
was
stripped
ol to
mechaed
fullanner
(22)
A
single RACE
product
was(Quik-Change
generated,
cloned
( TOPOa
quantitated
using
an
Imager
(Packard).
For
site-directed
mutagenesis
Site-Directed
or from
a wild-type
yeast strain
carrying
WT-�gluc
2% at
time 0.5%
0 andSDS
timeInstant
points
were
taken.
RNA was
with
boiling
and
probed
with
radioactively
ability
has
-,
gender-,
TA
2.1 kit,
Invitrogen),
and sequenced.
subcellular
fractionation,
transiently
transfected
Mutagenesis
Kit, Stratagene),
which
removed
the bona
plasmid
expressing
WT-PGK1
was used
as template.
extracted
and
analyzed
by northern
with
labeled
zeocin
cDNA. Northern
blotblotting
results
werea
ated CBP1
of
both(22)
the 16. G.
9,cloned
2421
(1995).
Caponigro,
Parker,
Genes
HeLa
cellsoligo
were
trypsinized,
washed
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andgenerated,
allDev.
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anner
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product
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labeled
probe
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(5�-CTCGGTCCTGquantitated
using
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Imager
(Packard).
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ition (21).
17.
A.
van
Hoof,
P.
A.
Frischmeyer,
H.
C.
Dietz,
R.
Parker,
phate
buffered
saline,
and
pelleted
by
centrifuging
at
UTR.
Ter-poly(A)-�gluc
was
generated
codons
in
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3�
TA 2.1 kit, Invitrogen), and sequenced.
TACCGAACGAGACGAGG-3�).
subcellular
fractionation, transiently transfected
xpression.
ated
CBP1
othesis
that
295, 2262
(2002).
Science
for 5were
min
at 4°C.
Cells
were
then
resuspended
2040g
via aGenes
single Dev.
point9,mutation,
which
from
nonstop-�gluc
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16.
G.
Caponigro,
R.
Parker,
32.
We
thank
N.
Martin,
A.
Atkin,
C.
Dieckmann,
and
M.
phosHeLa
cells
trypsinized,
washed
in
cold
1�
omprehenition
(21).
construct,
mouse
18.
create
theP.one
WT-�gluc
�l
140
mM and
NaCl,
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mM
in
200buffered
created
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codon minigene
upstream
the poly(A)
tail.
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forofthe
provision
of
valuable
reagents.
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17. To
A.
van
Hoof,
A. Frischmeyer,
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Dietz,
R. Parker,
of nonstop
phate
saline,
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by MgCl
centrifuging
at
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DNA
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used as by
template
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that
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295,
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for( supported
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at 4°C.
were
then
2040g
ction. The
�-glucuronidase
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three
different
portions
of
the
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on
ice
for
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min.
Nuclei
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quences
are
available
upon
request.
(H.C.D.),
the140
Howard
Hughes
Medical
Cs
by the 18. To create the WT-�gluc minigene construct, mouse
10 mM
mM NaCl,
1.5 mM
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in
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of
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19. from
P. Belgrader,
Cheng,
X. 3030
Zhou,
L. S. Stephenson,
(H.C.D, R.P.),
and
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Scientist
Progenomic
DNAJ. was
used
as template
for amplifying
Tris-HCl
( pH
8.6),the0.5%
and
T,
wction.
serves
as
The
and
part
of intron
1Biol.
(including
the(1994).
splice donorgene:
seand
subsequently
separated
from
aqueous
Maquat,
Mol.
Cell.
an phyloggram
(P.A.F.).
�-glucuronidase
three
different
portions
of14,the8219
vortexed,
and incubated
on ice
forthe
5 min.
Nuclei(cytowere
al
role
that
quence
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toCell.
13555
encompassplasmic)
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16,exon
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20. quence),
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Chasin,
Mol. encompassing
Biol.
from
nucleotide
to11285
3030
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pelleted by
centrifuging at 12,000g for 45 s at 4°C
olyadenyling
part
splice
acceptor
29. J.and
H.October
Graber, C.
R. Cantor,
S. C.from
Mohr,
F. 2002
Smith,(cytoProc.
(1996).
22
2001;
accepted
31
January
fidelity of
and
partofofintron
intron9 1(including
(includingthethe
splice
donor sesesubsequently
separated
theT.aqueous
an
pt phylogand its
quence
),
exon
10,
and
part
of
intron
10
(including
the
Natl.
Acad.
Sci.
U.S.A. 96, 14055 (1999).
quence),
from
nucleotide
11285
to
13555
encompassplasmic)
phase.
21. K. A. Sparks, C. L. Dieckmann, Nucleic Acids Res. 26,
aberrant
or
olyadenylsplice
donor
sequence),
and from
13556acceptor
to 15813
30.
Chen,
C. C.C.MacDonald,
J. Wilusz,
AcidsProc.
Res.
lation that
ing
part
of intron
9 (including
the splice
se29. F.
J. H.
Graber,
R. Cantor, S.
C. Mohr,Nucleic
T. F. Smith,
4676
(1998).
abundance
and
its
included
the
remainder
of intron
10,22
exon
11,
23,
(1995).
www.sciencemag.org
MARCH
2002
2261
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), exon SCIENCE
10,
part ofVOL
intron
10
(including
the
Natl.2614
Acad.
Sci. U.S.A. 96, 14055 (1999).
22. which
E. R. Oldham,
B. and
Bingham,
W.
R.295
Baumbach,
Mol.
nptproducts
intron
11,
and
exon
12.
All
three
fragments
were
TA
31.
pG-26
(25) containing
theNucleic
CBP1 Acids
gene Res.
unsplice
donor7,sequence),
and from 13556 to 15813
30. Plasmid
F. Chen, C.
C. MacDonald,
J. Wilusz,
ulation
that
Endocrinol.
1379 (1993).
or homeocloned
(
Topo
TA
2.1
kit,
Invitrogen),
sequenced,
and
der
the
control
of
the
GAL
promoter
and
the
LEU2
which
included
the
remainder
of
intron
10,
exon
11,
23, 2614 (1995).
23. C. Hilger, I. Velhagen, H. Zentgraf, C. H. Schroder,
ny products
nonstop
then
cloned
into
pZeoSV2
A single
basemarker
introduced
into wild-type
intron
11,65,
and
exon
12. All(Invitrogen).
three fragments
were
TA
31. selectable
Plasmid pG-26
(25)was
containing
the CBP1
gene un4284
(1991).
J. Virol.
mps allele was
or homeoto
gussequenced,
and
SKI7-deleted
Yeast
were grown
mecloned
( Topocorresponding
TA
2.1Cantor,
kit, Invitrogen),
and
der the
control ofstrains.
the GAL
promoter
and theinLEU2
24. pair
J. H.deletion
Graber,
C. R.
S. the
C. Mohr,
T. F. Smith,
generated
by
site-directed
mutagenesis
(Quik-Change
Nature
Struct.
dia
lacking marker
leucine and
galactose
at
by
then
cloned
into
pZeoSV2
A single baseselectable
was containing
introduced2%
into
wild-type
27, 888(Invitrogen).
(1999).
Nucleic
Acids
Res.
he nonstop
precise
the WTwas
pair
corresponding
the gusmpsofallele
25. Site-Directed
K. A.deletion
Sparks, Mutagenesis
S.
A. Mayer, Kit,
C.to
L.Stratagene)
Dieckmann,
Mol.
Cell.
of 0.5.Yeast
Cells were
were grown
pelleted
and
30°C
to an OD600strains.
and SKI7-deleted
in me-
z, Genes Dev.
transcripts (8). Half-lives were determined by plotting
s Dev. 8, 855
log plot. All half-lives were determined at 25°C except
for the ccr4� (performed at 30°C) and dcp1-2 [performed after shift to the nonpermissive temp (37°C) for
1 hour] experiments.
28. HeLa cells [maintained in DMEM with 10% fetal
bovine serum (FBS)] were grown to �80 to 90%
confluency, trypsinized, and resuspended in 300 �l of
RPMI 1640 without FBS, 10 mM glucose and 0.1 mM
dithiothreitol (DT T). Plasmid DNA (10 �g) was added
and cells were electroporated (Bio-Rad, Gene Pulser II
Electroporation System) at a voltage of 0.300 kV and
a capacitance of 500 �F. Poly(A) RNA (Oligotex midi
kit, Qiagen, 2 �g), isolated 36 hours after transfection, was separated on a 1.6% agarose formaldehyde
iencemag.org on June 29, 2007
ding
tran�gluc
construct
to create mutagenesis
nonsense-�gluc.
Nonstop990
nce 271,
resuspended
in media
lacking a 2%
carbon
source.
generated
by site-directed
(Quik-Change
dia lacking leucine
and containing
galactose
at
4199
(1997).
Biol. 17,
he mechaprecise
was generated
from WT-�gluc
twoofAcad.
rounds
of
Glucose
then added
to a final concentration of
Site-Directed
Mutagenesis
Kit, Stratagene)
the WTol
30°C to was
an OD
26. �gluc
A. Audibert,
M. Simonelig,
Proc. by
Natl.
Sci.
600 of 0.5. Cells were pelleted and
site-directed
mutagenesis
(Quik-Change
Site-Directed
nerated
from
2%
at time 0 and
time points
were
was
ding tran�gluc
create nonsense-�gluc. Nonstopresuspended
in media
lacking
a taken.
carbonRNA
source.
95, 14302to(1998).
U.S.A.construct
ability
has
Kit, Stratagene),
which
removed
the bona
ee point
muextracted
andthen
analyzed
blotting withof
a
�gluc
wasminigene
generated
from WT-�gluc
by two
of
Glucose was
addedbytonorthern
a final concentration
27. Mutagenesis
All PGK1
constructs
are under
the rounds
control
of
ol
mechaf both
the
fide
termination
codon
and
all
in-frame
termination
nesis
(Quiklabeled
oligo
probe
oRP1067
(5�-CTCGGTCCTGsite-directed
mutagenesis
(Quik-Change
Site-Directed
2%
at
time
0
and
time
points
were
taken.
RNA
was
the GAL1 upstream activation sequence (UAS). Cultures
ability
has
Ter-poly(A)-�gluc
was generated
codons
in theKit,
3�
, Stratagene)
TACCGAACGAGACGAGG-3�).
xpression.
Mutagenesis
Stratagene),
which
removed
the
bona
extracted and analyzed by northern blotting with a
were grown
to UTR.
mid-log
phase
in synthetic
complete
via containing
aand
single
which
from
nonstop-�gluc
nation
f bothcodon
the
32. We
thankoligo
N. Martin,
Atkin, C. (5�-CTCGGTCCTGDieckmann, and M.
fide
termination
codon
all point
in-frame
termination
labeled
probe A.oRP1067
media–uracil
(SC-ura)
2%mutation,
galactose
(tranomprehencreated
a
stop
one
codon
upstream
of
the
poly(A)
tail.
UTR.
the
3�
Sands
for the provision of valuable reagents. This
UTR.were
Ter-poly(A)-�gluc
was generated
codons
in on).
the 3�
TACCGAACGAGACGAGG-3�).
xpression.
scription
Cells
pelleted and resuspended
in
erated
deAll
changes
were
confirmed
by
sequencing.
Primer
secreated
from
work
was
supported
by
a
grant
from
NIH (GM55239)
via
a
single
point
mutation,
which
from
nonstop-�gluc
32. We thank N. Martin, A. Atkin, C. Dieckmann,
and M.
SC-ura media. Glucose was then added (transcription
omprehenquences
available
uponupstream
request.
directed
mu(H.C.D.),
Medical
Institute
Cs
by the
created
stop
one
codon
thealiquots
poly(A)were
tail.
Sands for the
the Howard
provision Hughes
of valuable
reagents.
This
off ) to aaare
final
concentration
of 2%ofand
19. P.
Belgrader,
J. Cheng,
X. Zhou,
L.points.
S. Stephenson,
E.
Mutagenesis
(H.C.D,
R.P.),
and thebyMedical
ProAll
changesatwere
confirmed
by sequencing.
PrimerL.sework was
supported
a grantScientist
from NIHTraining
(GM55239)
removed
the
indicated
time
Approximately
werated
servesdeas
14,
8219
(1994).
Maquat,
Mol.
Cell.
Biol.
firmed
by
segram
(P.A.F.).
quences
available
upon request.
(H.C.D.), the Howard Hughes Medical Institute
Cs
by that
the
total
RNA isolated
with the hot phenol meth10 �g ofare
l role
16,formal4426
20.
Kessler,
L.J. A.
Chasin,
Mol.
Biol.
ble
upon re19. O.
P.
Cheng,
X. Zhou,
S. Stephenson,
L. E.
(H.C.D, R.P.), and the Medical Scientist Training ProodBelgrader,
(4) was electrophoresed
on aL.Cell.
1.2%
agarose
wescribed
servesin of
as
(1996).
22
October
(8)
fidelity
14,
8219
(1994).
Maquat,
Mol.
Cell.
Biol.
gram
(P.A.F.).2001; accepted 31 January 2002
dehyde gel, transferred to a nylon membrane (Geneal role that
4426
20. O.
Kessler,
A. Chasin,
Mol. Cell.
Screen
Plus, L.NEN),
and hybridized
withBiol.
a 2316,
bp oligobertson,
Mol.
(1996).
22 October 2001; accepted 31 January 2002
fidelity of
nucleotide end-labeled radioactive probe that specifiwww.sciencemag.org
SCIENCE
295
22theMARCH
2002
2261
UTR of
PGK1
cally detects the
polyG tract VOL
in the 3�
2261
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VOL
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the percent ofSCIENCE
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versus
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semi-
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ev. 60, 233
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iol. 14, 8123
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23
connection
the innate
and adaptive
iDCs (Fig. 4C). Furthermore, pretreatment of further
Immunol. 169,
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3. Y. Wang
et al., J. between
K. T. Jean
(16), and
thetoxin
activation
and differentiation
269.May
4. M. J. responses
Gething, J. Sambrook,
Nature 355, 33 infection.
(1992).
during mycobacterial
iDCs
withcontrols
pertussis
or TAK-779
inhibited immune
J. E.2006;
Hildr
of Tability
cells (17).
Our finding that
a mycobacterial
10.1126/scien
5. P.cellular
Srivastava,aggregation
Annu. Rev. Immunol.
20, by
395 myHsp70
(2002).
Eur. J. Im
induced
the
of peptide-loaded
myHsp70
to stimu- The
late DCs to generate influenza peptide–specific signaling through CCR5 may play an important 10. P. Revy, M
Nat. Imm
CTLs (Fig. 4D). To examine the effect of CCR5- role in the formation of granulomas, the hallmark 11. M. L. Dus
than363
a lack
mediated signaling in mycobacterial infection, we of mycobacterial infection.
(200
of a
12. B. Kampm
Microbial-induced DC responses need to be cause
used the model pathogen M. bovis BCG-lux (12).
a Bod
sim
13. for
K. A.
As reported for murine DCs (13), human DCs highly regulated, reflecting a balance between a be
69, 800
is u
from immune people were unable to kill inter- rapid and appropriate response to invading mi- fitness
14. J. G. Cyst
nalized mycobacteria, even in the presence of crobes and the inducement of immunopathology 15. Coenzym
M. Nieto
1
1
1,2
genase
(IM
*
(2)—a
particular
problem
in
mycobacterial
inautologous
T
cells.
TAK-779
inhibition
of
CCR5
16. F. Castelli
Stephen P. Miller, Mark Lunzer, Antony M. Dean
etiology
and
17.
S.
A. Luth
led to a dose-dependent enhancement of intra- fection. An increasing number of human patho18. T.11).
Dragic
IM
gens,
including
HIV (18),
toxoplasma
(19), and (10,
cellular
4E andisfig.
The rolemycobacterial
of constraint replication
in adaptive(Fig.
evolution
an open
question.
Directed
evolution
of an engineered
19. J. Aliberti
of b
M. tuberculosis
(as described
here), target the ation
S6A)
at all concentrations
of mycobacteria
tested
b-isopropylmalate
dehydrogenase
(IMDH), with
coenzyme
specificity switched
from nicotinamide
20. E. de Silva
during
thegr
receptor.
This role(NADP),
of CCR5
as aproduces
pattern- 21. We are
(fig.
S6B),
suggesting(NAD)
an important
role for adenine
this CCR5
adenine
dinucleotide
to nicotinamide
dinucleotide
phosphate
always
acid.
Spectroph
receptor
for to
myHsp70
at least amino
receptor
controlling
mycobacterial
infection.
mutants in
with
lower affinities
for NADP.
This result is recognition
the correlated
response
selectionmay,
for relief
the Wellco
dinucleotide
in part,
be responsible
for thelandscape
maintenance
of the
The
identification
of CCR5
the critical
re-NADP)
from
inhibition
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to
(R.A.F.),
th
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high
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ceptor
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three enzymatic
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the rate
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maximum turnover
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in in ThisFEBS
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at
the
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European
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(20)constraints,
and may
implications
for both
mycobacterial
infectionbetween
and Northern
NADP and NADPH
affinities,
and a trade-off
NAD and NADP
usage.
Two additional
some relate
the patternprotein
of disease
seen have
in people
with Supporting
the
useNADPH
of myHsp70.
CCR5
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Figs.
S1 IDHs
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References and Notes
recruitment of cells to sites of inflammation (14),
Table
S1 use
enzyme
canJ. Chan,
be broken
by artificial
selection
heimmune
old notion
of natural
selection
as an constraint,
1. J. L. Flynn,
Trends Microbiol.
13, 98
(2005). to
facilitates
synapse
formation
(15), orchesVideos S1 to S
2. J. L. Flynn,
Chan, Annu. combinations
Rev. Immunol. 19,
939).
(2001).
replacemen
De- References
produce
new J.phenotypic
(8,
force of
in within
biological
evolution
trates Tomnipotent
lymph
nodes are
usually
opaque,
and
a
lack
of
response
hecell
oldinteractions
notion
natural
selection
3. Y. Wang et al., J. Immunol. 169, 2422 (2002).
all circumstantial evidence, results from Ile290 Y Ty
hascontrols
given way
to one where
adaptive pro- spite
(16), and
the activation
and differentiation
M. J. Gething,may
J. Sambrook,
355, 33
(1992).
reflectNature
nothing
more
than 26 May 2006;
as an omnipotent
force
in biologi- to4. selection
direct
experimental
tests
imply20,
that
cesses
are
constrained
by
physical,
chemical,
and
into the coe
of T cells
(17).
Our finding
that
a mycobacterial
5. P. Srivastava,
Annu. Rev.
Immunol.
395selection
(2002). is
a
lack
of
heritable
variation
(4,
7).
If
the 10.1126/scienc
cal
evolution
has
given
way
to
one
biological exigencies (1–4). Whether constraint largely unconstrained.
coli leuB–e
a constraint
is test
to be
where
adaptive processes
are constrained
The of
direct
experimental
for elucidated,
constraint is genesis cau
and/or stabilizing
selection explain
phenotypic cause
conceptually
simple.
A phenotype
is subjected
to than
stasis,
in the fossil
record and
phylogenies,
re- it
(10, 11):
N
must be for
a simple
phenotype
whose
by
physical,
chemical,
andinbiological
exia lack
selection (natural
or artificial)
in an attempt to cause
Direct experimental
mains an (1–4).
open question
(5). constraint
Km isofthe
to fitness
is understood.
gencies
Whether
and/or relationship
ac
the postulated
(6–9). A response be
tests of constraint
are scarce
(6–9).phenotypic
Even tight break
factor
34
for aofsim
Coenzyme
useconstraint
by β-isopropylmalate
stabilizing
selection
explain
correlations among traits, at once suggestive of to selection indicates a lack of constraint. No re- fitness
103 Mis–1un
stasis, in the fossil record and in phylog- dehydrogenase
(IMDH)
is
a
simple
pheNADP/K NA
sponse to selection indicates the presence of a (kcat
m
Coenzym
enies,
remains an1 open
question
(5). Direct notype
whose
etiology
and
constraint.
However,
the cause
of arelationship
constraint is genase
from 0.49
1
2
1
1,2
(IM
BioTechnology
Institute,Mark
Department
of Ecology,
Evolu*
Stephen
P.
Miller,
Lunzer,
Antony
M.
Dean
to
fitness
are understood
11). IMDHs
experimental
of constraint
scarce
rarely
specified
because the (10,
etiologies
of most etiology
The engine
and
tion and Behavior,tests
University
of Minnesota,are
St. Paul,
MN
phenotypesthe
are oxidative
not well understood,
their relationfinal11).
R IM
re
55108, USA.
catalyze
decarboxylation
of (10,
(6–9).
Even
tightin adaptive
correlations
among
The
role of
constraint
evolution
is an open
question. Directed
evolution
of an engineered
ships to fitness are usually
opaque,
and a lack of ation
wild-type
E
*To whom
correspondence
shouldofbeconstraint,
addressed. E-mail:
of
btraits,
at
once
suggestive
can
β-isopropylmalate
to
α-ketoisocaproate
b-isopropylmalate dehydrogenase (IMDH), with coenzyme specificity switched from nicotinamide
response to selection may reflect nothing more during
[email protected]
cific toward
the
be broken
by artificial
selection
to produce
the biosynthesis
of leucine,
an esadenine
dinucleotide
(NAD)
to nicotinamide
adenine during
dinucleotide
phosphate (NADP),
always produces
mutants
with lower combinations
affinities for NADP.
correlated
to selection
relief
new phenotypic
(8,This
9). result
De- is the
sential
aminoresponse
acid. All
IMDHsforuse
nico- amino acid.
dinucleotide
from
inhibition
by
NADPH
(the
reduced
form
of
NADP)
expected
of
an
adaptive
landscape
subject
20results
OCTOBER
2006 adenine
VOL 314dinucleotide
SCIENCE(NAD)
www.sciencemag.org
458 spite all circumstantial evidence,
tinamide
astoa This invaria
three enzymatic constraints: an upper limit to the rate of maximum turnover (kcat), a correlation in
from direct experimental tests imply that coenzyme (cosubstrate). This invariance of
NADP and NADPH affinities, and a trade-off between NAD and NADP usage. Two additional constraints, at the presen
selection
is largely
unconstrained.
among
IMDHs
hints
at ensured
the pres- some relate
high
intracellular
NADPH
abundance and the cost of function
compensatory
protein
synthesis,
have
directuseexperimental
testthroughout
for con-evolution.
ence Our
of results
ancientshow
constraints,
even
though use NADP
theThe
conserved
of NAD by IMDH
that selective
mechanisms
Structura
and
evolutionary
constraintssimple.
are to beAunderstood
terms ofrelated
underlying
adaptive landscapes.
isocitrate
dehydrogenases
straint
is conceptually
pheno- in some
using IDHs
(IDHs) use
instead
(12, 13).
type ishe subjected
(natural
canNADP
be broken
by artificial
selection to enzyme use
old notion to
of selection
natural selection
as or
an constraint,
artificial)
in an attempt
break the
pos- produce
Structural
comparisons
with(8,related
9). De- replacemen
new phenotypic
combinations
omnipotent
force in to
biological
evolution
NADP-using
IDHs evidence,
identify results
amino from
ac- Ile290 Y Ty
tulatedhasconstraint
response
to prose- spite
all circumstantial
given way(6–9).
to one A
where
adaptive
experimental coenzyme
tests imply that
is into the coe
cesses
constrained
physical,
chemical, No
and direct
lectionareindicates
a by
lack
of constraint.
ids controlling
useselection
(14–16)
unconstrained.
biological
(1–4).
Whetherthe
constraint
response exigencies
to selection
indicates
pres- largely
(Fig. 1A).
Introducing five replacements coli leuB–e
The direct experimental test for constraint is genesis cau
and/or stabilizing selection explain phenotypic
ence of a constraint. However, the cause of (Asp236 → Arg, Asp289 → Lys, Ile290→ Tyr,
simple. A phenotype
is subjected to (10, 11): N
stasis, in the fossil record and in phylogenies, re- conceptually
a constraint
rarely specified
the selection
Ala296→ (natural
Val, and
Gly337→ inTyr)
into the
or artificial)
an attempt
to Km is the
mains
an openisquestion
(5). Directbecause
experimental
etiologies
of mostarephenotypes
areEven
not well
coenzyme-binding
pocket (6–9).
of Escherichia
the postulated constraint
A response factor of 34
tests
of constraint
scarce (6–9).
tight break
selection
indicates aIMDH
lack of by
constraint.
No re- 103 M–1 s
correlations
at once suggestive
of to
understood,among
their traits,
relationships
to fitness
coli
leuB–encoded
site-directed
NADP/K NA
sponse
to selection
indicates
the presence
of a (kcat
m
mutagenesis
causes
a complete
reversal
constraint. However, the cause of a constraint is from 0.49 
1
2
in specificity (10, 11): NAD performance
BioTechnology Institute, Department of Ecology, Evolurarely
specified
because the etiologies of most The engine
NAD
tion and Behavior, University of Minnesota, St. Paul, MN
(kNAD
,where
Km is thetheirMichaecat /Km are
phenotypes
not well understood,
relation- final R rep
55108, USA.
lis
constant)
is
reduced
by aandfactor
ships to fitness are usually opaque,
a lack of
of wild-type E
*To whom correspondence should be addressed. E-mail:
3
response
to selection
[email protected]
340, from
68 × 10may
M–1reflect
s–1 tonothing
0.2 ×more
103 cific toward
Direct
Demonstration
of an
Direct Demonstration
of an
Adaptive Constraint
Adaptive
Constraint
T
T
Direct Demonstration of an
Adaptive Constraint
T
458 24
20 OCTOBER 2006
VOL 314
SCIENCE
www.sciencemag.org
M–1 s–1, whereas NADP performance
(kNADP
/KNADP
) is increased by a factor of
cat
m
70, from 0.49 × 103 M–1 s–1 to 34 × 103 M–1
s–1. The engineered LeuB[RKYVYR] mutant (the final R represents Arg341, already
present in wild-type E. coli IMDH) is as
active and as specific toward NADP as the
wild-type enzyme is toward NAD. Evidently, protein architecture has not constrained IMDH to use NAD since the last
common ancestor.
Despite similar in vitro performances,
the NADP-specific LeuB[RKYVYR] mu-
tant is less fit than the NAD-specific wild
type (11). IMDHs, wild-type and mutant
alike, display a factor of 30 higher affinity
for the reduced form of NADP (NADPH)
(coproduct) than for NADP (Fig. 1B). We
suggested the LeuB[RKYVYR] mutant is
subject to intense inhibition by intracellular NADPH, which is far more abundant
in vivo than is NADP (11, 17). The inhibition slows leucine biosynthesis, reduces
growth rate, and lowers Darwinian fitness
(Fig. 1C). The wild type retains high fitness because its affinity for NADPH is
REPORTS
Fig. 1. The molecular anatomy of an adaptive constraint.
(A) Structural
) seen with engin
and NADPH
(KiNADPH
phosphorus)
with labels
designating
the
alignment of the coenzyme binding pockets of E. coli amino
IMDH acid
(14)and
(brown
in theinscreened
mutants (dots). (C) Th
site number
IMDH followed
main chain) with bound NAD modeled from Thermus thermophilus
IMDH
(11) showing
by the aminoIMDH
acid inbyIDH.
Coenzyme
use the phenotype
in chemostat
and descr
(15) and E. coli IDH (16) (green main chain) with bound
NADP. Only by
keyH bonds
is determined
to NAD competition
(brown
residues are shown (gray, carbon; red, oxygen; blue,lines)
nitrogen;
predicted
distribution
and to yellow,
the 2’-phosphate
(2’P)
of NADP of performanc
phosphorus) with labels designating the amino acid and
sitelines).
number
in binding
pocket
(gray dots), with wil
(green
(B) Mutations
in the
coenzyme
IMDH followed by the amino acid in IDH. Coenzyme use binding
is determined
by that
H dot),
and NADP
single also
amino acid repla
pocket
stabilize
of NADP
(greenThebinding
bonds to NAD (brown lines) and to the 2¶-phosphate (2¶P)
(pink dots). The fitne
stabilize
NADPH.
ensuingpocket
correlation
NADP
lines). (B) Mutations in the coenzyme binding pocket that
thanNADPH
in the wild type bec
stabilize for
NADP
) and
in affinities
NADP be
(Kmlower
NADP)
NADPH
intracellular
NADPH
also stabilize NADPH. The ensuing correlation in affinities(Kfor
NADP
(K
)
seen
with
engineered
mutants(11).
m
i
(circles) (11) is retained in the screened
toward NAD. Evidently, protein architecture has
mutants (dots). (C) The adaptive landscape
not constrained IMDH to use NAD since the last
for coenzyme use by IMDH (11) showing
common ancestor.
the phenotype-fitness map (blue surface)
Despite similar in vitro performances, the
determined in chemostat competition and
NADP-specific LeuBERKYVYR^ mutant is less
described by equation S5 (11, 18) and the
fit than the NAD-specific wild type (11). IMDHs,
predicted distribution of performances
NADPH
) seen
with
engineered
mutants
(circles)
retained
DPH (Ki
Fig. 1.
The
molecular
anatomy
ofa factor
an (11)
adaptive
for 512 mutants in the coenzyme binding
wild-type
and
mutant alike,
display
ofis30
creened mutants
(dots).
(C) (A)
The
landscape
constraint.
Structural
alignment
of the use
pocket (gray dots), with wild type (red dot),
higher
affinity
for adaptive
the reduced
form for
of coenzyme
NADP
H (11) showingcoenzyme
the phenotype-fitness
mapof(blue
surface)
determined
binding pockets
E. coli
IMDH
(14)
LeuB[RKYVYR] (black dot), and single amino
(NADPH) (coproduct)
than for
NADP
(Fig. 1B).
mostat competition
and
described
by
equation
S5
(11,
18)
and
the
main
with bound NAD
modeled
acid replacements in LeuB[RKYVYR] coenzyme
We(brown
suggested
thechain)
LeuBERKYVYR^
mutant
is subed distribution from
of performances
for 512 mutants
in the
Thermus
thermophilus
IMDH (15)
andcoenzyme
E.
binding pocket (pink dots). The fitness of
ject
to
intense
inhibition
by
intracellular
NADPH,
pocket (gray dots),
with(16)
wild(green
type (red
dot),
LeuB[RKYVYR]
coli is
IDH
main
chain)
with
bound (black
LeuB[RKYVYR] is hypothesized to be lower than
which
far
more
abundant
in
vivo
than
is
NADP
nd single amino
acid Only
replacements
in LeuB[RKYVYR]
coenzyme
NADP.
key residues
are shown
(gray,
in the wild type because of strong inhibition by
17).The
Thefitness
inhibition
slows leucine
biosynthesis,
pocket (pink(11,
dots).
of
LeuB[RKYVYR]
is
hypothesized
to
carbon; red, oxygen; blue, nitrogen; yellow,
abundant
intracellular NADPH (11)
reduces
growth
rate,
and
lowers
Darwinian
fitness
er than in the wild type because of strong inhibition by abundant
(Fig.
1C).
The
wild
type
retains
high
fitness
because
Fig. 2. The phenotypic basis of the genetic screen. Lowered expr
lular NADPH (11).
25 increasing co
its affinity for NADPH is low, whereas inhibition by brings IMDH to saturation with isopropylmalate,
NADH, which is far less abundant than NAD in expression in the chemostat-derived adaptive landscape [lower con
H. Coenzyme use is determined by H
2¶-phosphate (2¶P) of NADP (green
binding pocket that stabilize NADP
lation in affinities for NADP (KmNADP)
dot), and single amino acid replacements in LeuB[RKYVYR] coenzyme
binding pocket (pink dots). The fitness of LeuB[RKYVYR] is hypothesized to
be lower than in the wild type because of strong inhibition by abundant
intracellular NADPH (11).
ecture has
ce the last
nces, the
ant is less
. IMDHs,
ctor of 30
of NADP
(Fig. 1B).
ant is subNADPH,
is NADP
osynthesis,
ian fitness
ss because
hibition by
n NAD in
quence of
ture (18),
ADPH inuse.
trained to
associated
r, identifyinhibition)
causes of
DP (maxibreak the
nities (Fig.
coenzyme
enefit the
promising
therefore
use NAD
NADP, an
cat
of NADP
ade-off in
Fig. 2. The phenotypic basis of the genetic screen. Lowered expression in the presence of excess glucose
brings IMDH to saturation with isopropylmalate, increasing coenzyme affinities. (A) Lowering IMDH
expression in the chemostat-derived adaptive landscape [lower concentration of E in equation S5 (11, 18)] is
predicted to reduce the fitness of LeuB[RKYVYR] (white sphere) far more than that of the wild type (red
sphere). (B) IPTG-controlled expression of IMDHs ligated downstream of the T7 promoter in pETcoco in strain
RFS[DE3] (leuAþBamCþ, with T7 RNA polymerase expressed from a chromosomal lacUV5 promoter) confirms
that lower expression affects growth in minimal glucose medium of the LeuB[RKYVYR] (white) more than in
the wild type (red). Plasmids lacking LeuB (black) are incapable of growth except in the presence of leucine.
low,
inhibition
by (targeted
NADH, random
which
Wewhereas
used directed
evolution
mutagenesis
selective
screening)
to
is far less and
abundant
than
NAD in(19–21)
vivo, is
test
whether NADP-specific
with higher
ineffective.
Perhaps as aIMDHs
consequence
of
fitness
could beinisolated.
Random
substitutions
differences
Michaelis
complex
strucwere introduced into leuB[ RKYVYR] by means
ture
(18),
IDH
is
not
subject
to
such
inof error-prone polymerase chain reaction (18).
tense NADPH
inhibition
and hence could
Mutated
alleles were
ligated downstream
of the
NADP
use.
T7evolve
promoter
in pETcoco
(a stable single-copy
vector)
transformedthat
into IMDH
strain RFSEDE3^
We and
hypothesize
is conþBamCþ, with T7 RNA polymerase ex(leuA
strained
to use NAD because the strong
pressed
from associated
a chromosomal
inhibition
withlacUV5
NADPpromoter).
use reSequencing unscreened plasmids revealed that,
duces fitness. However, identifying the
on average, each 1110–base pair leuB[ RKYVYR]
mechanism
of selection
(NADPHFrom
inhibireceived
two nucleotide
substitutions.
the
tion) of
is base
not synonymous
pattern
substitutions in with
these identifying
mutants and
ing) (19–21)
to statistics,
test whether
NADP-speassuming
Poisson
we estimate
that only
10.5
of the
2431higher
possiblefitness
amino could
acid recific (0.4%)
IMDHs
with
placements
wereRandom
missing insubstitutions
our mutant library
(18).
be isolated.
were
Our experimental
design usedbydecreased
introduced
into leuB[RKYVYR]
means
IMDH expression to provide a simple selective
of error-prone polymerase chain reaction
screen for mutations in leuB[ RKYVYR] that in(18). Mutated
alleles
wereAs
ligated
downcrease
growth and/or
fitness.
predicted
from
stream
of the T7 promoter
in pETcoco
(a
the
phenotype-fitness
map (Fig.
2A), selection
against
leuB[ RKYVYR]
intensified
as IMDH
stable single-copy
vector)
and transformed
expression
lowered (leuA
in the +presence
of excess
into strainwas
RFS[DE3]
BamC+, with
T7
glucose
(Fig. 2B). Using
40 mMfrom
isopropyl-bRNA polymerase
expressed
a chro-Dthiogalactopyranoside (IPTG) to induce a low
mosomal lacUV5 promoter). Sequencing
level of IMDH expression, we found that cells
unscreened
plasmids
revealed
on avharboring
leuB[
WT] formed
largethat,
colonies
at 24
erage, each
1110–base
pair leuB[RKYVYR]
hours,
whereas
cells harboring
leuB[ RKYVYR]
the causes of constraint. Mutations that received two nucleotide substitutions.
increase kNADP
(maximum rate of NADP From the pattern of base substitutions in
cat
www.sciencemag.org SCIENCE VOL 314 20 OCTOBER 2006
459
turnover), that break the correlation in these mutants and assuming Poisson statisNADP and NADPH affinities (Fig. 1B), tics, we estimate that only 10.5 (0.4%) of
or that eliminate the trade-off in coen- the 2431 possible amino acid replacements
zyme performances (Fig. 1C) could each were missing in our mutant library (18).
Our experimental design used debenefit the LeuB[RKYVYR] mutant without compromising its performance with creased IMDH expression to provide a
NADP (18). We therefore hypothesize simple selective screen for mutations
that IMDH is constrained to use NAD by in leuB[RKYVYR] that increase growth
three causes: an upper limit to kNADP
, an and/or fitness. As predicted from the
cat
unbreakable correlation in the affinities of phenotype-fitness map (Fig. 2A), selecNADP and NADPH, and an inescapable tion against leuB[RKYVYR] intensified
trade-off in coenzyme performance.
as IMDH expression was lowered in the
We used directed evolution (targeted presence of excess glucose (Fig. 2B).
random mutagenesis and selective screen- Using 40 μM isopropyl-β-D-thiogalacto26
replacement in the coenzyme binding pocket, or
both. Upstream substitutions occurred at three
nucleotide positions: –3, –9, and –14 relative to
eliminate H bonds to the 2¶-phosphate of NADP
to cause striking reductions in NADP performance
(Table 1). Although some mutants have improved
Table 1. Kinetic effects of amino acid replacements in LeuB[RKYVYR] isolated at 48 hours growth.
Standard errors are G13% of estimates. Single-letter abbreviations for amino acid residues: A, Ala;
C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R,
Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr.
NAD
NADP
Enzyme
Km
(mM)
Site
DDIAGR (wild type)
RKYVYR
Performance
(A)
kcat
kcat/Km
(s–1) (mM–1 s–1)
8400 4.10
183 6.20
Km
(mM)
Performance Preference KiNADPH
(B)
(A/B)
(mM)
kcat
kcat/Km
(s–1) (mM–1 s–1)
0.4900
101 6.90
68.3200
33.8800
4108 0.83
0.2000
Beneficial replacements
K235N
3919 5.95
1.5200
6356 0.50
0.0800
K235N,–3(M),A60V
1744 5.06
2.9000
5395 0.44
0.0800
K235R
554 6.39
11.5000
3292 0.36
0.1100
K235R,E63V
658 6.75
10.3000
3748 0.46
0.1200
K289E*
3449 2.71
0.7900
4897 1.98
0.4000
K289E*,D317E
3554 3.62
1.0200
5580 2.09
0.3700
K289E*,D87Y,S182F
2551 2.54
1.0000
5750 3.65
0.6300
K289E*,F102L
3891 3.66
0.9400
4823 1.10
0.2300
K289M
2216 4.13
1.8600
4034 0.48
0.1200
K289M,F170L
1945 2.50
1.2900
2947 0.48
0.1600
K289N*
1141 6.32
5.5400
4997 1.14
0.2300
K289N*,R187H
1146 5.83
5.0900
3069 0.79
0.2600
K289N*,H367Q
1248 5.27
4.2200
4555 1.05
0.2300
K289T
1596 6.18
3.8700
4056 0.78
0.1900
K289T,Q157H
1696 5.66
3.3400
5038 0.93
0.1800
Y290C
988 5.21
5.2700
3792 1.02
0.2700
Y290C,R152C
910 3.15
3.4600
3844 0.90
0.2300
Y290D
10213 3.13
0.3100
9040 0.82
0.0900
Y290F*
1658 5.59
3.3700
3110 0.65
0.2100
Y290F,P97S,D314E
1097 4.64
4.2300
4158 0.71
0.1700
Y290N,N52T
2381 3.44
1.4400
10660 0.26
0.0200
Replacements with beneficial 5¶-leader mutations
RKYVYR
183 6.20
33.8800
4108 0.83
0.2000
–3(M)†,E66D
208 2.97
14.2800
5002 0.30
0.0600
–3(M)†,E66D,E331D
267 7.99
29.9000
3403 0.34
0.1000
E82D
221 5.44
24.6200
4735 0.35
0.0700
K100R,A229T,L248M
137 5.58
40.3700
4079 0.45
0.1100
G156R,K289R,Y311H
292 5.40
18.4900
4464 0.54
0.1200
E173D
219 6.39
29.1800
5304 0.89
0.1700
Y337N,G131
171 5.71
33.3900
3367 0.24
0.0700
Y337H,P85S,E181K
318 5.21
16.3800
2409 0.22
0.0900
0.007
169.400
254.30
9.90
19.000
35.700
106.100
84.100
2.000
2.800
1.600
4.100
15.500
8.100
24.300
19.600
18.300
20.400
18.600
19.600
14.800
3.400
16.000
24.800
59.500
129.00
56.00
26.00
28.00
133.50
112.00
105.20
131.90
78.60
87.20
44.00
44.70
57.80
71.60
72.60
49.00
43.00
344.00
59.70
57.40
90.00
169.400
238.000
296.900
351.700
370.300
144.800
171.600
468.500
183.600
9.90
11.90
14.00
14.10
11.50
17.40
6.60
8.00
12.80
*Switch from NADP to NAD requires two base substitutions in one codon. This is a possible transitional amino acid produced by
†The –3(M) designates a G to A substitution at base –3 that creates a new start codon.
a single base substitution (11).
460
are benef
formance
if there ar
upper lim
for NAD
enzyme p
with redu
phenotyp
remains f
performan
of NADP
cial as co
NADPH f
As predi
mutants h
and NAD
increases
eficial (18
that NAD
vivo beca
NADPH.
and increa
the predic
map cons
correlation
and a trad
Break
NADP-sp
increases
for NADP
trade-off
clusions a
is too stri
distinct m
limit, 17
leader seq
LeuBERK
ments in
zyme pe
inhibition
cial muta
teria dem
not overly
Nor ar
sampling
adaptive
sequential
lution de
mutants
coli are n
force mu
vantageo
pyranoside (IPTG) to induce a low level terion compatible with reliably identifying
20 OCTOBER 2006 VOL 314 SCIENCE www.sciencemag.or
of IMDH expression, we found that cells beneficial mutations in leuB[RKYVYR].
harboring leuB[WT] formed large colo- Longer periods of growth (or higher connies at 24 hours, whereas cells harboring centrations of IPTG) allowed unmutated
leuB[RKYVYR] barely formed pinprick leuB[RKYVYR] to form colonies, whereas
colonies at 48 hours. We chose colony for- shorter periods (or lower concentrations of
mation at 48 hours as the least stringent cri- IPTG) produced no colonies.
27
Of the 100,000 mutated plasmids
screened, 134 (representing 107 distinct
isolates) formed colonies within 48 hours
(table S1). Each had either a substitution
in the 5′ leader sequence upstream of
leuB[RKYVYR], an amino acid replacement in the coenzyme binding pocket, or
both. Upstream substitutions occurred at
three nucleotide positions: –3, –9, and –14
relative to the leuB AUG start codon (fig.
S1). Ten amino acid replacements were
found at three codons in the coenzyme
binding pocket.
At first glance, the positive response to
selection might suggest that coenzyme use
by IMDH is unconstrained. Upstream substitutions in the Shine-Dalgarno sequence
(positions –9 and –14), as well as a new
AUG start codon (position –3) that replaces the less efficient GUG start codon,
presumably derive their benefits through
increases in expression because their kinetics are unchanged. Unexpectedly, however, beneficial amino acid replacements
in the coenzyme binding pocket eliminate
H bonds to the 2′-phosphate of NADP to
cause striking reductions in NADP performance (Table 1). Although some mutants
have improved NAD performance, others
remain unchanged and several show reduced NAD performance. Isolated amino
acid replacements outside the coenzyme
binding pocket, which might have been
expected to increase kNADP
or to break the
cat
correlation in affinities between NADP
and NADPH (KNADP
and KNADPH
), have
m
i
no detectable functional effects (table S2,
those associated with beneficial 5′-leader
mutations in Table 1). No doubt they,
along with 126 silent substitutions (table
S1), hitchhiked through the genetic screen
with the beneficial mutations.
Our results suggest that increases in expression are beneficial, whereas increases
in NADP performance are not. This seeming paradox is resolved if there are no
mutations capable of breaking the upper
limit to kNADP
the correlation in affinities
cat
for NADP and NADPH, or the trade-off in
28
coenzyme performance. With these constraints, and with reduced expression in
the genetic screen, the phenotype-fitness
map near LeuB[RKYVYR] remains flat
with respect to increases in NADP performance (Fig. 2A). By contrast, severe losses of NADP performance are predicted to
be beneficial as correlated reductions in
the affinities for NADPH free up IMDH
for use with abundant NAD. As predicted,
all beneficial LeuB[RKYVYR] mutants
have reduced affinities for both NADP and
NADPH (Table 1). Unaffected by constraints, increases in expression are unconditionally beneficial (18). These results
support the hypothesis that NADP-specific
IMDHs function poorly in vivo because of
strong inhibition by abundant NADPH.
That reductions in NADP performance
and increases in expression are both beneficial are the predicted consequences of a
phenotype-fitness map constrained by an
upper limit to kNADP
, a correlation in affinicat
ties for NADP and NADPH, and a tradeoff in coenzyme performance.
Breaking any one constraint would allow NADP-specific IMDHs to evolve.
Yet no mutant increases kNADP
, no mutant
cat
uncouples the affinities for NADP and
NADPH, and no mutant breaks the tradeoff in coenzyme performance. These conclusions are not the result of a selective
screen that is too stringent. Of 35 colonies,
representing 26 distinct mutants, that appeared after the 48-hour limit, 17 had no
nucleotide substitutions in the 5′ leader
sequence or amino acid replacements in
LeuB[RKYVYR] (table S3). Amino acid
replacements in the other mutants neither improved enzyme performance nor
decreased NADPH inhibition (table S4).
That no additional beneficial mutations
were recovered with relaxed criteria demonstrates that the selective screen was not
overly stringent.
Nor are the results a consequence of
inadequate sampling of protein sequence
space. Natural adaptive evolution fixes
advantageous mutations sequentially (22–
ampling of
volutionary
.04 advanre missing
ry (18). We
ampling
of
reaking the
volutionary
are unlikely
.04
advanse they
are
re
t), missing
because
yboth.
(18). We
eaking
the
an NADPre
ar unlikely
NADPH
se
are
asethey
exprest),
because
relieves
the
both.
sources of
an
theNADPrest of
ar
NADPH
enefit
to be
ase
expres(Fig.
1C)
the
selieves
such that
sources
of
above
wildthe rest the
of
ercome
enefit
to be
g resources
1C)
rd(Fig.
compense such
that
a protein
bove
wildthe evoluercome the
g resources
notypes,
by
rd compenering,
is not
e constraints
a protein
the
evoluconstraints
be identified
otypes,
by
phenotype,
ring,
is not
landscape.
constraints
used
to test
,constraints
we have
elationships
e phenotype
24). Indeed, experimental evolution demonstrates that advantageous double mutants in the evolved ß-galactosidase of E.
coli are not evolutionarily accessible and
perforce must be accumulated as sequential advantageous mutations (25). Hence,
screening all single substitutions is a
sufficient sampling of protein sequence
space for robust evolutionary conclusions.
We estimate that only 0.04 advantageous
amino acid replacements are missing from
the mutant leuB[RKYVYR] library (18).
We conclude that mutations capable of
breaking the limit, the correlation, or the
trade-off are unlikely to ever be fixed in
populations because they are exceedingly
rare (they may not exist), because they are
minimally advantageous, or both.
The two remaining ways to evolve an
NADP-specific
IMDH
are tocan
reduce
intraunderlying
a conserved
phenotype
be identified
cellular
NADPH
pool
and,
as
our
results
from the relationships among genotype, phenotype,
and
fitness
that define
an adaptive
landscape.
show,
to increase
expression.
Reducing
inExperimental
evolution relieves
can then the
be used
to test
tracellular NADPH
inhibition
their
Using this
approach,
we have
but, existence.
as experiments
deleting
of
underlying
conserved
phenotype
cansources
be
identified
shown howa certain
structure-function
relationships
NADPH
show
(13),
the
disruption
to
the
from
the
relationships
among
genotype,
phenotype,
in IMDH have constrained its coenzyme phenotype
and
defineancestor.
an adaptive
rest fitness
of
costs
far morelandscape.
than the
since
themetabolism
lastthat
common
Experimental evolution can then be used to test
their existence. Using this approach, we have
References
andstructure-function
Notes
shown
how certain
relationships
S. J. Gould, R. C. Lewontin, Proc. R. Soc. London Ser. B
in1.IMDH
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8.
11.
9.
12.
10.
13.
benefit to be gained. The phenotype-fitness
map (Fig. 1C) imposes a law of diminishing returns such that LeuB[RKYVYR]
must be expressed above wild-type levels
by a factor of 100 to overcome the inhibition by NADPH (18). Diverting resources
away from other metabolic needs toward
compensatory protein synthesis would impose a protein burden (26–29) sufficient
to prevent the evolution of NADP-specific
IMDHs.
The production of unnatural phenotypes, by artificial selection or molecular
engineering, is not sufficient to conclude
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identified from the relationships among
REPORTS
genotype, phenotype, and fitness that
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Experimental
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tion
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25.
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September
26S2006;
proteasome
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10.1126/science.1133479
context, drastic knockdown of p75 changed the
genomic pattern of HIV-1 integration by reducing
the viral bias for active genes, which suggests that
29
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rapid degradation
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Tech Notes
Large Insertions: Two Simple Steps Using
QuikChange® II Site-Directed Mutagenesis Kits
Abstract
This Technical Note describes a modified
method to introduce large insertions in
plasmids in two simple steps using the
QuikChange® II Site-Directed Mutagenesis Kit. Dr. Wenge Wang in Dr. Wafik S.
El-Deiry’s lab at the University of Pennsylvania has used the QuikChange kit and
a modification of the method described
by Geiser et al. (2001)1 to perform a large
insertion of a PCR product into a target
plasmid. He had inserted the enhanced
green fluorescent protein (EGFP) open
reading frame (760 bp) following the Cterminus of death receptor TRAIL receptor 1/death receptor 4 (TRAIL-R1/DR4)
right after the signal sequence, using the
QuikChange II site-directed mutagenesis
kit with some modifications to the basic
protocol. Dr. Wang transfected cells with
the recombined plasmid and it generated
a fusion protein with the correct molecular
size (Figure 1).
Background
Dr. Wafik S. El-Deiry’s lab is focused on
studying the mechanism of action of the
tumor suppressor p53 and the contribution of its downstream target genes to
control cell growth. Their lab has identified a number of genes that are directly
regulated by p53 and which can inhibit
cell cycle progression (p21WAF1), induce
apoptosis (KILLER/DR5, Bid, caspase 6,
Traf4 and others) or activate DNA repair
(DDB2). The research has provided knowledge into the tissue specificity of the DNA
damage response in vivo and into the
mechanism by which wild-type p53 sensitizes cells to killing by anti-cancer drugs.
High-Efficiency Mutagenesis Method
All QuikChange® kitsa offer a one-day
method to introduce point mutations, amino acid substitutions, small insertions, and
deletions in virtually any double-stranded
30
plasmid template at efficiencies greater
than 80% (Figure 2). The QuikChange II
and QuikChange II XL kits feature our
highest fidelity DNA polymerase, our gold
standard for accuracy, and a linear amplification strategy. Together, they reduce the
mutation frequency due to incorporation
errors typically caused by using an exponential PCR amplification approach. The
result is high-efficiency mutagenesis without unwanted errors.
Ideal for Large Insertions and Deletions
We have used the QuikChange kit method
for small insertions and deletions (~12 bp)
and observed greater than 80% efficiency2.
Many of our customers have used the
QuikChange method or have modified it
to introduce deletions of 31 bp3, 87 bp4,
and 3 kb5 or insertions of 31 bp3 and up
to ~1 kb5. In order to achieve large insertions of up to 1 kb, megaprimers must be
generated from an initial PCR reaction. To
ensure the highest mutagenesis efficiency,
we recommend gel purification with our
StrataPrep® PCR Purification Kit. To create
the megaprimers, a minimum of 20 bp
upstream and downstream of the insertion
sequence should be complementary to
your template vector that will be used in
the QuikChange kit reaction. Therefore,
each oligo used in the initial PCR reaction will require 20 bp overhangs on the
5’ ends.
Using the QuikChange kit for large deletions is even easier since oligos can be
synthesized, rendering the initial PCR reaction and fragment purification unnecessary. Again, 20 to 30 bp may be required
to bind to your template DNA both upstream and downstream of the region that
you want to delete. However, the largest
oligo required should not exceed 60 bp.
This Technical Note describes performing
a large insertion that can be completed
in two simple steps. First, PCR-amplify
the megaprimer. Second, add your gel
purified megaprimer to our QuikChange
site-directed mutagenesis kit (Figure 2).
This strategy avoids tedious and time-consuming sub-cloning. With our QuikChange
kits, you will have confidence in generating an error free clone while saving valuable time.
Materials & Methods
Part I. PCR reaction to generate
the megaprimer
Reaction and cycling conditions.
PCR reagents: PfuUltra® II Fusion HS DNA
Polymerase (Stratagene)
Thermal Cycler used: TECHNE: Touchgene® Gradient
• The forward primer: GACTCCGAATCC
CGGGAGCGCAGCGGTGAGCAAGG
GCGAGGAG, the first 25 bp overlaps
the target vector, the remaining primer
sequence is complementary to EGFP.
The primers were resuspended in 50
mM NaCl.
• The reverse primer: CGCGGCTGCCTC
TGTCCCACTCTTGTACAGCTCGTCCA
TGCC, the first 22 bp targets the vector
and the remaining primer sequence is
complementary to EGFP. The primers
were resuspended in 50 mM NaCl.
• Template for PCR reaction: 200 ng of
plasmid DNA (pEGFP-N1; Clontech)
•
PfuUltra® II Fusion HS DNA Polymerase (Stratagene)
• PCR product was purified with
QIAGEN’S QIAEX II Gel Extraction Kit
• The quantity of the PCR product was
estimated on agarose gel with Ethidium
Bromide
Fig. 1 Enhanced green fluorescent protein (EGFP)
was inserted in the open reading frame (760 bp) following the C-terminus of death receptor TRAIL receptor 1/death receptor 4 (TRAIL-R1/DR4) right after the
signal sequence. Cell staining shows the member
protein fused with EGFP.
1. Mutant Strand Synthesis
Perform thermal cycling to:
• Denature DNA template
• Anneal mutagenic primers
containing desired mutation
• Extend and incorporate primers
with high-fidelity DNA polymerase
2. Dpn I Digestion of Template
Digest parental methylated and
hemimethylated DNA with Dpn I
3. Transformation
Transform mutated molecule into
competant cells for nick repair
Fig. 2. The QuikChange® II One-Day Site-Directed
Mutagenesis Method. 1. Mutant strand synthesis. 2.
Dpn I Digestion of parental DNA template. 3. Transformation of the resulting annealed double-stranded
nicked DNA molecules. After transformation, the XL-1
Blue E. coli cell repairs nicks in the plasmid.
Note: The size of the PCR product is 760
bp, which includes the insert (EGFP) of
714 bp plus overlapping sequence of the
target vector.
31
Tech Notes
Part II. The QuikChange II
mutagenesis reaction can
be set up using the following guidelines (Tables 1
and 2).
• Primer for the insertion reaction 300-500
ng of PCR product (760
bp EGFP) to serve as a
megaprimer.
Note: Follow reaction
with Dpn I digestion and
transformation per the
QuikChange® II manual.
For PfuUltra® II Fusion HS
DNA Polymerase information please refer to
the manual.
TABLE 1. Reaction Set-up
Amount
Component
per reaction
Distilled water (dH2O)
X µl
10x QuikChange reaction buffer
5.0 µl
dNTP mix
1 µl
DNA template (50 ng)
X µl
Megaprimer (300 - 500 ng)
X µl
High Fidelity DNA polymerase (2.5 U/µl)
1.0 µl (2.5U)
Total reaction volume
50 µl
TABLE 2. Cycling Method
Segment Cycles
1
1
2
5
3
13
Temperature
Time
95°C
30 seconds
95°C
30 seconds
52°C
1 minute
68°C
1 min/kb of
plasmid* (7 min)
95°C
30 seconds
Results
55°C
1 minute
The results of this mu1 min/kb of
68°C
tagenesis experiment
plasmid* (7 min)
demonstrate that large
* For example, a 7 kb plasmid would have a 7 minute extension time
PCR fragments can be successfully inserted into the target plasmid DNA. The 760 bp PCR fragment had at
each end only short regions of homology where the insertion occurred. There were
approximately 100 colonies produced. About 20 colonies were selected for further
analysis of mutagenesis efficiency, and they were 100% positive by sequencing.
Acknowledgments
This Technical Note was written based on data provided by Wenge Wang, M.D.,
Ph.D., in Dr. Wafik S. El-Deiry’s laboratory, Division of Hematology and Oncology in
the Department of Medicine at the University of Pennsylvania.
References
1. Geiser, M., et al. (2001) BioTechniques 31:
88-92.
2. Papworth, C., Braman, J., and Wright, D.A.
(1996) Strategies 9: 3-4.
3. Wang, W. and Malcolm, B. (1999) BioTechniques 26: 680-682.
4. Burke, T., et al. (1998) Oncogene 16: 1031-1040.
5. Makarova, O., et al. (2000) BioTechniques 29:
970-972.
Legal
QuikChange® and StrataPrep® are registered
32
trademarks of Stratagene, an Agilent Technologies company.
QIAGEN® is a registered trademark of QIAGEN.
Touchgene® is the registered trademark of Techne Incorporated.
Clontech© logo is the trademark of Clontech
Laboratories, Inc.
a. U.S. Patent Nos. 7,176,004; 7,132,265;
7,045,328; 6,734,293; 6,489,150; 6,444,428;
6,391,548; 6,183,997; 5,948,663; 5,932,419;
5,866,395; 5,789,166; 5,545,552, and patents
pending.
  
With GeneMorph II mutagenesis kits, a balanced spectrum of mutations is right at your fingertipss
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     
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Our GeneMorph II Kits include Mutazyme II
Polymerase, which delivers a balanced mutational
spectrum.
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GC to N
     
      
     
Mutazyme II
DNA Polymerase
®
       
Mutazyme I
DNA Polymerase
®
      
Taq
DNA Polymerase
      
Value
10% 20% 30% 40% 50% 60% 70% 80%
      
        
 
       
  
   
    
 

     
 


u.s. and canada 1-800-424-5444 x3
europe 00800-7000-7000
www.stratagene.com
U.S. Patent No. 7,045,328; 6,803,216; 6,734,293; 6,489,150; 6,444,428; 6,183,997; 5,489,523 and patents pending
GeneMorph and Mutazyme are registered trademarks of Stratagene, an Agilent Technologies Company,
in the United States.
®
®


  
2X Faster
     

    
      

 
 


        
    
 
      
 
 
    



 
       


     
     

       
u.s. and canada 1-800-424-5444 x3
europe 00800-7000-7000
www.stratagene.com
QuikChange is a registered trademark of Stratagene, an Agilent Technologies Company, in the United States.
*U.S. Patent Nos 6,734,293; 6,489,150; 6,444,428; 6,391,548; 6,183,997; 5,948,663;
5,932,419; 5,866,395; 5,789,166; 5,545,552; 6,713,285 and patents pending.
®
      
    

  
  