THE JOURNAL OF BIOL.OCKXL CHEMHTRY
Q 1990 by The American Society for Biochemistry
Vol. 265, No. 25, Issue of September 5, pp. 15145-15153,199O
Printed in U.S.A.
and Molecular Biology, Inc.
Transcription
Pausing by Escherichia coli RNA Polymerase
Modulated by Downstream DNA Sequences*
(Received
Donna
N. Lee$,
Le PhungSP,
From the Departments
of SBiology
St. Louis, Missouri
63130
Judith
Stewart&
and llBiochemistry
and Robert
and Molecular
Escherichia
coli RNA polymerase
pauses immediately after transcription
of certain
sequences
that can
form stable secondary
structures
in the nascent
RNA
transcript;
pausing
appears
to be essential
for several
types of bacterial
transcription
attenuation
mechanisms. Because
base changes
that weaken
the RNA
secondary
structures
reduce the half-life
of pausing
by
RNA polymerase,
nascent transcript
RNA hairpins
are
thought
to cause pausing
at these sites. We show here
that, for the well characterized
trpL
pause site, the
determinants
of transcription
pausing
are not limited
to the RNA hairpin,
but include
the not-yet-transcribed
sequence
of DNA immediately
downstream
from the
pause site. We show that this effect extends to bases up
to fourteen
nucleotides
downstream
from the pause
site, that placement
of a oligo(dT)
tract in the nontranscribed strand in this region does not convert
the pause
site to a termination
site, and that shifting
the position
of pausing
by one nucleotide
downstream
almost eliminates pausing.
From an analysis
of many variants
of
this downstream
sequence,
we argue that the effect of
downstream
sequence
is not related
simply
to its GC
content.
We suggest that these effects are mediated
by
altered
interactions
between
RNA polymerase
and the
DNA template
downstream
from the enzyme’s
active
site.
Transcription by Escherichia coli RNA polymerase is discontinuous (l-4). At most positions on the template, the
enzyme rapidly catalyzes RNA chain elongation; at certain
“pause” sites, however, RNA polymerase requires 100-1000
times longer to extend the RNA chain (5). Two types of pause
sites have been described: (i) those that occur immediately
after a region of dyad symmetry that can produce an RNA
hairpin in the nascent transcript, termed RNA hairpin-induced pausing (3, 5), and (ii) those that occur where no
potential secondary structures are obvious, termed sequencedependent pausing (l-3, 5). The most notable RNA hairpinassociated pause sites occur in the leader regions of amino
acid biosynthetic operons that are regulated by attenuation.
Here, transcription pausing is thought to play a key role in
uiuo, by stopping the transcribing RNA polymerase until a
* This work was supported
by Grant GM-38660
from the National
Institute
of General
Medical
Science,
a Presidential
Young Investigator Award
from the National
Science
Foundation,
and an award
from the Searle Scholars
Program.
The costs of publication
of this
article
were defrayed
in part by the payment
of page charges.
This
article
must therefore
be hereby
marked
“advertisement”
in accordance with 18 USC.
Section
1734 solely to indicate
this fact.
§ Current
address:
Dept. of Biochemistry
and Molecular
Biology,
The University
of Chicago,
Chicago,
Illinois
60637.
(1 To whom correspondence
should be addressed.
Is
for publication,
April
13, 1990)
LandickSliII
Biophysics,
Washington
University,
ribosome initiates translation of the leader peptide coding
region.
The requirement of a stable, base-paired RNA structure for
transcription pausing has been inferred from analyses of the
effect on pausing of sequence changes in the DNA segments
that specify the RNA hairpin (6,7). The mechanism by which
formation of a nascent transcript RNA hairpin causes the
transcription complex to stop elongation is unknown. Available evidence is consistent with the view that the putative
RNA hairpin structure forms within the transcription complex and disrupts at least a portion of the RNA:DNA heteroduptex (8). At least three substantial controversies remain:
(i) do RNA hairpins interact directly with RNA polymerase
to alter its activity (9) or do they affect elongation simply by
disrupting a portion of the RNA:DNA heteroduplex (5, 10);
(ii) are pause RNA hairpins fundamentally different from
termination RNA hairpins or do they simply disrupt less of
the RNA:DNA heteroduplex; and (iii) is the RNA:DNA hybrid 12 f 1 base pairs, as conventionally thought (5), or is a
substantial portion of the RNA in the transcription complex
actually bound in a nascent transcript binding site (ll)?’ If
the recently synthesized RNA actually lies in such a binding
site, then models in which RNA hairpin formation partially
or completely removes it from this site or in which direct
RNA-RNA polymerase interactions influence pausing also
should be considered. To more completely characterize the
causes of transcription pausing, we have studied the effect on
pausing of DNA sequences downstream from the pause site.
We show here that a complete description of transcription
pausing in the trp leader region cannot be limited solely to
the causes and effects of nascent transcript RNA hairpin
formation. Not-yet-transcribed DNA sequences downstream
from the catalytic site in trpL paused transcription complexes
alter the half-life of the complex. Specifically, we show that
replacement of these sequences with arbitrarily chosen sequences reduces the pause half-life by a factor of 3 and that
this reduction does not correlate with the GC content of the
replacement sequence. This effect extends at least 14 nucleotides past the site of pausing, roughly to the downstream
border of RNA polymerase on DNA in the paused transcription complex (8). We argue that this effect arises from the
interaction of RNA polymerase with the double-stranded
DNA helix in the portion of the transcription complex downstream from the catalytic site.
EXPERIMENTAL
Materials,
Bacterial
Strains,
PROCEDURES
and DNA
Manipulations-E.
coli RNA
polymerasewas prepared by the method of Burgessand Jendrisak
(12). NusA
Chamberlin
bonucleotides
15145
’ C. Kane
protein
was purified
by the method
of Schmidt
and
(13) from an overproducing
E. coli strain
(14). Deoxyriwere from Sigma; high performance
liquid chromatogand M. Chamberlin,
personal
communication.
15146
Downstream
DNA
Sequences
raphy-purified
ribonucleotides
were purchased
from Pharmacia
LKB
Biotechnology
Inc. Plasmid
DNAs
were routinelv
isolated
bv the
alkaline
lysis-procedure
(15) from strain
RL511.
RL5ll
(also designated KTl)
is a derivative
of HBlOl
(16) that carries
an unknown
mutation
that stabilizes
plasmids
containing
strong
E. coli RNA
polymerase
promoters.*
DNA
fragments
for in vitro transcription
reactions
were prepared
by the polymerase
chain reaction,
using the
M13-20
universal
primer
(New England
Biolabs)
and a custom
oligonucleotide
(5’.d[GCTTCGCAACGTTCAAATCC]-3’)
that paired
with a sequence
in rrnB
Tl T2 DNA to amplify
the desired
DNA
fragments
directly
from -200
ng of plasmid
DNA
(17). The DNA
fragments
were recovered
from polymerase
chain reactions
by extraction with phenol/chloroform
followed
by ethanol
precipitation.
The
fragments
were dissolved
in TE buffer
(10 mM Tris-HCI,
1 mM
Na,EDTA,
pH 7.9) and used directly
for in vitro transcription
by E.
coli RNA polymerase
(see below).
DNA sequencing
was performed
by the dideoxynucleotide
sequencing
method
(18,
19)
using
modified
T7
DNA
polymerase
(SequenaseTM,
United
States
Biochemical
Corp.),
[a-35S]dATP
(Amersham
Corp.),
and synthetic
oligonucleotide
primers.
Singlestranded
DNA
for sequencing
was prepared
by polymerase
chain
reaction
amplification
of the DNAs of interest
using the Ml3 universal primer that had been modified
to contain
biotin at the 5’ end (20)
and a custom oligonucleotide
that matched
a sequence
in rrnB Tl T2
DNA.
The biotin-tagged
DNAs
were bound to strepavidin-agarose,
loaded on top of a Sephadex
G-50 column,
and strand-separated
with
NaOH
as described
bv Mitchell
and Merril
(21). The collected
DNA
was ethanol-precipitated
and used directly
in the DNA sequencing
reactions.
Plasmid
Constructions-Plasmids
pRL407,
pRL417,
and pRL424
are related to the pUC119
(22) derivative
pRL418
(7), which contains
the phage T7 Al promoter
from pAR1707
(23), a short polylinker,
and the rrnB Tl T2 terminator
region (24). pRL407
contains
a RsaI
(trpL
+ 44) to TaqI (trpL
+ 187) fragment
from the wild-type
trp
leader region in the HincII
site of pRL418.
pRL417
contains
a RsaI
(trpLep
+ 44) to NlaIII
(trpLep
+ 97) fragment
of the trpLep derivative of the trp operon’
between
the HincII
and SphI sites of pRL418.
pRL424
contains
a RsaI (trpLep
+ 44) to Sau3A
(trpLep
+ 260;
treated
with
DNA
polymerase
I Klenow
fragment)
fragment
of
trplep”
between
the X&I
and PstI sites of pRL418
that had been
treated with T4 DNA polymerase
to produce
blunt ends.
pRL433
was derived
by oligonucleotide-directed
mutagenesis
(26)
of pRL417
and contains
an NcoI site at the trpL transcription
pause
site (Table
I). ~RL456.
~RL457.
and pRL458
were derived
from
pRL433
by insertions
of double-stranded,
synthetic
oligonucleotides
in the NcoI site (Table
I). pRL459
and pRL520
were unintended
artifacts
that arose from these procedures
(Table I). pRL491,
pRL492,
pRL496,
pRL497,
and pRL499
were derived
similarly
from pRL433
using self-complementary
oligonucleotides
that specified
various
homopolymer
or alternating
polymer
tracts
(Table
I). pRL500
was
similarly
derived
from pRL433
using oligonucleotides
that restored
39 nucleotides
of wild-type
sequence
after the trpL pause site followed
by nucleotides
that specify
XhoI,
HpaI, BglII, and StuI restriction
endonuclease
recognition
sites (Table I).
~RL553
and aRL498
were constructed
by insertion
of synthetic,
double-strandedoligonucleotides
between
the unique NcoI and BglII
sites in pRL538.
pRL538
is a derivative
of pRL500
that contains,
in
the DNA polymerase
I Klenow
fragment-modified
NcoI site, a forward-oriented
BamHI
to SphI fragment
from the tet gene of pBR322
(27) that had been modified
to make the BamHI
site blunt-ended
and
to add to the SphI site a synthetic,
double-stranded
DNA sequence,
AGATCTGCTAG
d ( GTACTCTAGACGATC
) .
The deletion
series pRL509
through
pRL519
was prepared
by
exonuclease
III/mung
bean nuclease
(Stratagene)
treatment
of XhoIcleaved
pRL500
following
a protocol
supplied
by the vendor.
Nuclease-treated
DNAs
were recovered
by phenol
extraction
and
ethanol
precipitation
and
ligated
to synthetic
XhoI
linkers
(dlCTCGAGCTCGAG1).
These samples then were treated with XhoI
and BamHI
and electrophoresed
through
low-melting
agarose.
Fragments
of the desired
sizes were excised
from the gel and directly
ligated pRL500
that had been cut with BamHI
and XhoI. Plasmids
L
1.
’ J. Majors,
personal
communication.
” Landick,
R., Yanofsky,
C., Choo,
Biol., in press.
K., and Phung,
L. (1990)
J. Mol.
Modulate
Transcription
Pausing
derived
by this procedure
were screened
for the size of the BumHIXhoI fragment
and promising
candidates
were sequenced.
In Vitro
Transcription
Reactions-Standard
synchronous
transcription
reactions
were performed
at 37 “C essentially
as described
previously
(9). A20 complexes
(28) were formed
on the polymerase
chain reaction-synthesized
DNA fragments
by incubation
of 2.5 pmol
of RNA polymerase
and 1 pmol of DNA in 50 ~1 of 40 mM Tris-HCl,
20 mM NaCl,
14 mM MgCl,,
14 mM P-mercaptoethanol,
2% (v/v)
glycerol,
20 pg of acetylated
bovine serum albumin/ml,
240 pM ApU
dinucleotide,
and 2.5 MM ATP, CTP, and GTP for 20 min at 37 “C.
Elongation
from A20 was initiated
by adjusting
GTP to 20 FM and
the ATP. CTP. and UTP to 150 uM. Five-u1
aliauots
were removed
at appropriate
time intervals
and mixed with 5 ~1 of 2 X TBE, 0.025%
bromphenol
blue, 0.025%
xylene
cyan01 saturated
with urea. RNA
samples
were analyzed
by electrophoresis
through
0.4 mm x 24 cm X
30.cm 10% polyacrylamide,
7 M urea gels in TBE buffer. Radioactivity
in the pause RNA bands was quantitated
by placing
the gel in an
AMBIS
radioanalytic
scanner
and using algorithms
supplied
by the
manufacturer.
After
subtraction
of appropriate
background
values,
these data were used for semi-logarithmic
regression
analysis
and the
pseudo-first
order rate of pause RNA disappearance
was determined
from the slope.
RESULTS
Replacement of Sequences Downstream from the trpL Pause
Site Lowered the Pause Half-life by a Factor of 3-We
discovered the effect of downstream
sequence on transcription
pausing at the trpL pause site during analysis of in vitro transcription of the altered trp leader region from plasmid pRL417
(Fig. 1; Table I). On pRL417, the DNA sequence immediately
after the trpL pause site was replaced by sequences from the
E. coli rrnB terminator
region (rrnZ3 Tl T2; see Ref. 24).
When this truncated
trpL DNA was transcribed
in uitro by
E. coli RNA polymerase, the half-life of pausing at the trpL
pause site was reduced from the wild-type
level of 44 s
observed with pRL407, to 13 s (Figs. 1 and 2; Table I). The
reduction
in pausing by a factor of 3 also occurred when
transcription
was conducted in the presence of NusA (Fig. 2,
C and D; Table I).
In this initial comparison,
the wild-type
trpL region fused
to the T7 Al promoter
was compared to a truncated
pause
site DNA that contained a minor alteration to the loop of the
pause RNA hairpin and is designated trpLep (Fig. 1). Analyses
of wild-type
trpL and trpLep under a variety
of conditions
have revealed no difference in any transcriptional
phenotype
either in uiuo or in vitro (29, 30).3 To verify that the effect on
pausing that we observed was caused by differences in the
downstream
sequence and not by the difference in the pause
RNA hairpin loops, we prepared and tested a T7 Al-trpLep
fusion, pRL424, which contains the trpLep pause RNA hairpin and wild-type attenuator
and differs from pRL417 only
downstream
from the pause site (Table I). This new plasmid
gave the same pause half-lives
as pRL407 with or without
NusA (Table I). Thus we concluded that the reduction by a
factor of three in the pause RNA half-life
on the pRL417
template was due solely to differences
in the not-yet-transcribed DNA downstream from the pause site. On the pRL417
template, the reduction in pause half-life was not caused by
the first three nucleotides after the pause site, since these are
the same in pRL407, pRL417, and pRL424.
Properties of the Template DNA Molecule Ztself within 35
Nucleotides
Downstream
from the Pause Site Modulate
the
Half-life of Transcription
Pausing in the trp Leader-To
better
delineate the effect of downstream DNA sequence on pausing,
we prepared
and tested several additional
derivatives
of
pRL417. To rule out the possibility
that reduced pausing on
the pRL417 template was due to the absence of DNA sequences from the trpL termination
region, we constructed and
tested pausing on pRL455 and pRL458, derivatives that lack
Downstream
DNA Sequences Modulate
Transcription
15147
Pausing
A
B
PpL
77 Al - frpL FUSION
1:2
WA
G
U
A
A
C
C,GG
C&G
FIG. 1. Schematic
representation
of transcription
templates
and sequence
of
transcripts
from
the
pRL407
and pRL417
templates.
A,
arrangement
of transcription
template
components
(not to scale).
Details
of
construction
of the templates
are given
under
“Experimental
Procedures.”
PCRl
and PCR2 designate
the approximate positions
at which oligonucleotides
hybridize
for polymerase
chain reaction
amplification
of the transcription
templates. B, sequence
of the pRL407
transcript up to the trpL terminator.
Bases
shown
in bold are derived
from the T7
Al transcription
unit and polylinker
of
plasmid
pUC19
(25). Arrows
indicate
the
position
of pausing
by E. coli RNA polymerase.
C, sequence
of the pRL417
transcript
up to the rrnB Tl terminator.
u
AUCGAG
c
g:;
A=lJ
CrG
ii
G
Gto
WG
C
G
A
t?
70
AAU
U
c
AG
IIOC=G
G=C
C=Gm
C’-G
C=G
G=C
uA
UU
GX
U=A
GzC
GICSO
GGGAUCCUCUAGAGUCAC~GAAAGGUU=AUGCGUAAAGCAAUCAGAGACCCA=UUUUUUUU
:
G
20
40
4
PAUSE
C
9o
la
l&J
rrnB
T7 Al - trpLep - rrnB FUSION “$,,JP
..
Tl
AUCGAG
$
G
G
10
t
A
C
G
E
&GAUCC~JCUAGACUGCAG~~P
20
30
g”c
C=G
A=U
U”
GEC
U=A
GEC
GrC
rGGUU =AUGCGAGAGUAGGGAACUGCCAGGAUCAAAG
ids’”
A=U
U=A
A= U
110
@-=4
m
‘w
PAUSE
the 34 terminator region, but which contain wild-type sequence for 35 nucleotides downstream from the pause site
(Table I). These two plasmids are identical except that
pRL455 contains two repeats of the downstream DNA sequence, whereas pRL458 contains only a single copy of this
sequence (Table I). Pausing on the pRL455 and pRL458
templates was indistinguishable from wild type (Table I).
Thus the effect of downstream DNA sequence on trpL pausing
was not mediated by formation in the DNA of the structure
analogous to the 3:4 termination hairpin.
To determine whether or not the exact wild-type DNA
sequence downstream from the pause site was required for
normal pausing, we prepared two single base substitutions.
Substitution of a C for the T at position +4 (pRL457) or a C
for an A at position +6 (pRL456) did not reduce significantly
the pause half-life (Table I). Thus, normal transcription pausing at the trpL pause site does not require an exact downstream DNA sequence; single substitutions at positions +4
and +6 have minimal effects on the half-life of trpL paused
transcription complexes.
To test if the effect of downstream sequence on pausing
was somehow related to binding of nucleotide triphosphate
substrates, we examined pausing on the pRL433 template,
which specifies 3 tandem G residues immediately after the
pause site. Pausing in the trp leader is sensitive to the concentration of GTP (31). This alteration also created an NcoI
15148
Downstream
DNA Sequences Modulate
Transcription
Pausing
TABLE I
Half-lives
of transcription
complexes
paused at the trpL pause site on downstream
sequence variants
Synchronized
transcription
reactions
at 20 KM GTP with or without
50 nM NusA protein
at 37 “C were performed
as described
under
“Experimental
Procedures.”
Half-lives
were determined
as described
under
“Experimental
Procedures.”
Two standard-deviation
error was calculated
from the regression
analysis.
Designation
Sequence
Half-life
transcription
after pause site”
of paused
complexes
-NusA
+NusA
s
pRL407
pRL417
pRL424
pRL458
pRL455
pRL456
pRL457
pRL433
GCGTAAAGCAATCAGATACCCAGCCCGCCTAATGAGGCGGGCTTTTTTTT(wild-type)
GCGAGAGTAGGGAACCTGCCAGGCATCAAAGAAAACGAAAGGCACAGTCG
GCGTAAAGCAATCAGATACCCAGCCCGCCTAATGAGGCGGGCTTTTTTTT(trpLep)
GCGTAAAGCAATCAGATACCCAGCCCGCCTAATGAGATCTGTTAACTCGA
GCGTAAAGCAATCAGATACCCAGCCCGCCTAATGAGATCTGTTAACTCGAGCCAT~
GCGTACAGCAATCAGATACCCAGCCCGCCTAATGAGATCTGTTAACTCGA
GCGCAAAGCAATCAGATACCCAGCCCGCCTAATGAGATCTGTTAACTCGA
GGGAGAGTAGGGAACCTGCCAGGCATCAAAGAAAACGAAAGGCACAGTCG
pRL553
pRL511
pRL512
pRL513
pRL514
pRL515
pRL509
pRL510
GGCATTTCGTTAGTCTATGGGTCGGGCGGATTACAGATCCTCTACGCCGG
GCGCTCGAGTTAACAGATCTAGGCCTTCGAG
GCGTCTCGAGTTAACAGATCTAGGCCTTCGAG
GCGTACTCGAGTTAACAGATCTAGGCCTTCGAG
GCGTAACTCGAGTTAACAGATCTAGGCCTTCGAG
GCGTAAAGCTCGAGTTAACAGATCTAGGCCTTCGAG
GCGTAAAGCCTCGAGTTAACAGATCTAGGCCTTCGAG
GCGTAAAGCACTCGAGTTAACAGATCTAGGCCTTCGAG
GCGTAAAGCAACTCGAGTTAACAGATCTAGGCCTTCGAG
GCGTAAAGCAATCTCGAGTTAACAGATCTAGGCCTTCGAG
GCGTAAAGCAATCCTCGAGTTAACAGATCTAGGCCTTCGAG
GCGTAAAGCAATCAGACTCGAGTTAACAGATCTAGGCCTTCGAG
GCGTAAAGCAATCAGATACCCAGCCCGCCTAATGAGATCCTCGAGTT~CAGAT
GTTTTTTTTCATGGGAGAGTAGGGAACCTGCCAGGCATCAAAGAAAACGA
GAAAAAAAACATGGGAGAGTAGGGAACCTGCCAGGCATCAAAGAAAACGA
GATATATATCATGGGAGAGTAGGGAACCTGCCAGGCATCAAAGAAAACGA
GCCCCCCCCCATGGGAGAGTAGGGAACCTGCCAGGCATCAAAGAAAACGA
GGGGGGGGCGATCTGCTAGCATGGGAGAGTAGGGAACCTGGCCAGGCATC
GCGCGCGCGCATGGGAGAGTAGGGAACCTGCCAGGCATCAAAGAAAACGA
CGCACAGCAATCAGATACCCAGCCCGCCTAATGAGATC
CGTACAGCAATCAGATACCCAGCCCGCCTAATGAGATC
pRL516
pRL517
pRL518
pRL519
pRL500
pRL491
pRL492
pRL496
pRL497
pRL498
pRL499
pRL520
nRL459
(trpLep
A3:4)
(trpLep
A4)
44 f 4
13 t 2
42 -t 4
50+ 15
43 + 4
51 c9
41 +- 4
15 + 3
140
32 f
140
ND*
150 +
146 k
156 +
35 *
32 + 6
41+ 4
26 + 3
22+ 11
29 f 2
50 -c 8
43 f 6
50 f 5
52 f 7
60 f 9
59 -+ 8
40f
10
48 + 6
18 f 3
16 + 2
10 + 2
16 f 3
10 + 3
26 f 4
<5‘+
<5
78 +
130 +
89 +
82 +
80 f
120 k
93 k
140 t
155 f
230 +150 k
ND
115 f
50 +
32 f
22 +
52 k
32 _c
115 +
<5
<5
7
26
50
37
11
13
16
14
14
18
12
8
34
15
16
75
21
10
15
15
8
6
23
’ Italicized
sequences
are those that differ from the wild-type
sequence.
Some sequences
have been underlined
to emphasize
certain
aspects of the altered sequences.
’ ND, not determined.
’ pRL455
differs
from pRL458
in that the sequence
from +3 to the end on that shown is repeated
once.
d Pausing either was not detectable
or was too short to allow half-life
determination.
We estimate that 5 s is the
maximum
half-life
for pausing
that would not be detectable
in these assays.
site that was useful for further manipulations
(Table I; see
“Experimental
Procedures”).
The pause half-life
of trpL
paused transcription
complexes formed on the pRL433 template was identical to that of complexes formed on the pRL417
template (Table I). That a requirement
for three G residues
in a row immediately
after the pause did not increase the
pause half-life
during in vitro transcription
at 20 FM GTP
suggests that the effect of downstream
DNA sequence on
pausing is not due to binding of the nucleoside triphosphates
specified by the sequence but to properties
of the DNA molecule itself. This conclusion
is supported further by the observation of extremely weak pausing on a variant template
that specifies 8 consecutive Gs after the pause site (pRL498,
Table I; see below).
To determine whether the orientation
of base pairs after
the pause site contributes
to the effect of downstream
sequence on pausing, we tested the effect of inverting each base
pair after the pause site so that the overall composition
and
order of base pairs was maintained,
but the sequence of the
two DNA strands was reversed (pRL533; Table I). We observed a slight, but significant,
reduction in pause half-life on
the pRL533
template
(Table I), suggesting that the base
sequence of each DNA strand and not simply the order of
base pairs, contributes to the effects of downstream
sequence
on transcription
pausing.
Alteration
Site Affects
of Sequences
Transcription
up to 14 Nucleotides
past
Pausing-To
determine
the
Pause
how far
downstream
from the pause site the effect of DNA sequence
on pausing extends, we prepared and tested a set of derivatives
of pRL458 (trpLep
plus 35 nucleotides
of wild-type
downstream DNA sequence; Table I) that altered progressively
greater amounts of this DNA (Table I; Fig. 3). To facilitate
construction
of these altered templates, we used a XhoI-linker
sequence to replace the wild-type sequence downstream of the
trpL
pause site. When this sequence was placed at +4, in a
position analogous to the sequence substitution
in pRL417,
the pause half-life was unaffected
(pRL511; Table I, Fig. 3).
However, as the amount of wild-type sequence was increased,
Downstream
A
pRL407
DNA Sequences Modulate
Transcription
Pausing
15149
NusA
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-.
P
-
-a-
w*.m
i
1
7
3
I:
j
6
-
3
0
‘3
‘2
3
‘t
AUGCGTAAAGCAA-CAGATAC*****
POSITION
OF XHOI-LINKER
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PAUSE CTCGAGTTAACAGAT
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--0
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-*rryryIc
T
P‘
---
AI
FIG. 2. Autoradiograms
from
gels containing
pause
RNAs
from
transcription
of the pRL407
and pRL417
templates.
Synchronized
transcription
reactions
with or without
50 nM NusA
protein
at 37 “C were performed
as described
under “Experimental
Procedures.”
Samples were removed
from the reactions
at the times
indicated
above the lane designations.
A, pRL407
(wild-type
trpL)
template
without
NusA. Lane 1, sample removed
prior to addition
of
chase mix t.o reaction;
lane 2, sample
removed
after 10 s; lane 3,
sample removed
after 20 s; lane 4, sample removed
after 30 s; lane 5,
sample removed
after 45 s; lane 6, sample removed
after 1 min; lane
7, sample removed
after 1.5 min; lane 8, sample removed
after 2 min;
lane 9, sample removed
after 2.5 min; lane IO, sample removed
after
3.5 min. The positions
of the A20 (A), pause (P), and terminated
(ZJ
RNAs are indicated
on the left. B, pRL417
(trpLep:rrnR) template
without
NusA. Samples
in each lane and markers
on left are as for A.
C, pRL407
(wild-type
trpL) template
with 50 nM NusA.
Lane 1,
sample removed
prior to addition
of chase mix to reaction;
lane 2,
sample removed
after 30 s; lane 3, sample removed
after 1 min; lane
4, sample removed
after 1.5 min; lane 5, sample removed
after 2 min;
lane 6, sample removed after 2.5 min; lane 7, sample removed after
3.5 min; lane 8, sample removed
after 5 min; lane 9, sample removed
after 7 min; lane 10, sample removed
after 12 min. Markers
on left
are as for A. D, pRL417
(trpLep:rrnB) template with NusA. Samples
in each lane and markers
on left are as for C.
the pause
half-life
fell significantly
and was roughly twothirds of the wild-type level when six nucleotides of wild-type
sequence were present (pRL514; Table I, Fig. 3). We did not
obtain derivatives with the linker sequence attached to the
7th or 8th nucleotide downstream
from the pause site, but
when the XhoI-linker
sequence was attached to nucleotides
9, 10, 11, or 13, we observed a slightly greater than wild-type
pause RNA half-life
(pRL509-pRL518;
Table I, Fig. 3). Fi-
5
‘C
I’
b
‘I
ATTACHMENT
SEQUENCE SEPLACING
W 7 AT IND CA-ED
?OSI-IOIS
FIG. 3. Pause half-life
uersus the position
of sequence
alteration downstream
from
the trpL pause
RNA hairpin.
Data are
from Table
1. The wild-type
sequence
downst.ream
from the trpL,
pause site and last two bases of the trpL pause RNA are shown below
the graph. The replacement
sequence
is shown
below it. Half-lives
are plotted
above the position
at which
the replacement
sequence
would begin in the various
constructs.
Note that, since cytosine
is
normally
present
at position
9, pRL515
alters the wild-type
sequence
only from position
10 on; thus the data from pRL515
and pRL509
were averaged and plotted at position
10. The same is true for position
13 and pRL517
and pRL518;
the averaged
data are plotted
at position
14.
nally, with 16 nucleotides of wild-type sequence present, the
pause RNA half-life returned to the wild-type level (pRL519;
Table I, Fig. 3). We conclude that nucleotides up to at least
position +14 make some contribution
to setting the half-life
of transcription
pausing in the trp leader.
Substitution
of Homopolymer
Sequences Downstream from
the Pause Site Suggests That GC-richness Does Not Determine
the Contribution
of Downstream
Sequences to Transcription
Pausing-Several
researchers
who have investigated
sequence-dependent
pausing have suggested that GC-rich DNA
could slow the transcription
complex by presenting
a barrier
to unwinding
the duplex (Refs. 5, 32, 33; see “Discussion”).
To determine
the role of downstream
sequence composition
in determining
the trpL paused transcription
complex halflife, we prepared and tested variants of pRL433 in which
eight-nucleotide
homopolymer
tracts were placed from +2 to
+9 relative to the pause site, in the region that we found to
be important
for the downstream
sequence effect. We found
that any tract of eight identical
or alternating
nucleotides
significantly
reduced the pause half-life
(pRL491-pRL499;
Table I). The strongest reductions
occurred with a run of
eight dG nucleotides or four (dA-dT) dinucleotides
(half-lives
= 10 s; pRL498 and pRL496, Table I). Significantly,
the
presence of three different
arrangements
of eight C-G base
pairs did not increase the pause RNA half-life to above the
wild-type
level (pRL497-pRL499;
Table I). Moreover, a run
of 8 Cs in the nontranscribed
strand (pRL497) gave a reduced
pause RNA half-life that was identical to that produced by a
run of eight Ts or eight As (pRL491 and pRL492; Table I).
Clearly, the GC content of the DNA sequence downstream
from the trpL pause site is not the primary determinant
by
which these sequences influence transcription
pausing.
Placement of a dT Tract Immediately
Downstream Does Not
Convert the trpL Pause Site to a Termination
Site-An
oftencited model for p-independent
termination
is that formation
of the secondary structure in the nascent transcript
induces
RNA polymerase to pause and that subsequent synthesis of
oligoribo(U)
allows release of the transcript,
presumably
be-
15150
Downstream
DNA Sequences Modulate
cause the rU-dA base pairs do not form a sufficiently stable
RNA:DNA hybrid (5, 9). From this view, one particularly
interesting variant that we obtained was pRL491, which contains a run of eight T residues immediately after the trpL
pause site. This modification did not cause any transcription
termination at the run of Ts; all transcription complexes
transcribed past this sequence to the end of the DNA template
either at 20 FM GTP or at 2.5 pM GTP and 50 nM NusA
protein (Fig. 4, C and F), conditions that should maximize
termination (6). Thus a simple test of the notion that a pindependent termination site is composed of an RNA hairpininduced pause site followed by a run of T residues is negative.
However, we believe that a complete test of this view requires
systematic variation of the position of the T-tract relative to
the segments that specify the RNA hairpin. As yet, we have
not completed such an analysis.
The Effects of Downstream
Sequence
on Pausing Are Not
Mediated by the NusA Transcription
Factor-For each of the
variants we obtained, we determined the pause RNA half-life
in the presence and absence of 50 nM NusA protein (Table
I). In general, we observed a 3-fold increase in pause half-life
in the presence of NusA, regardless of what the half-life was
without NusA. Thus the contribution of downstream sequence
to pausing most likely is independent of the NusA transcription factor. These findings are consistent with the view that
NusA interacts with and stabilizes the RNA hairpin in a
paused transcription complex (8).
A
pRL455
20$4 GTP N”r/\
B
pRL459
20 IIt.4GTP N”sA
C pRL491
20)iMGE=. N”sA
Transcription
Pausing
Shifting the Position of Pausing One Nucleotide Drastically
Reduces Its Half-Life-Two
interesting derivatives of pRL458
that arose from an artifact in the cloning procedures (pRL459
and pRL520) rearranged the downstream DNA sequence so
that the G nearest the pause site was one nucleotide further
away from the pause RNA hairpin. The site of pausing on
these templates at limiting GTP was shifted one nucleotide
further downstream, as would be predicted from the sequence
(verified by high-resolution electrophoresis; data not shown).
However, transcription pausing was almost eliminated on
these templates (Table I; Fig. 4, B and E). This effect was
much greater than any we observed from variations in the
downstream sequence alone, and thus is not likely to be due
to the sequence downstream from the new pause site but to
the altered position of pausing. Even at 2.5 FM GTP and 50
nM NusA protein, the pause half-life on the pRL459 and
pRL520 templates was less than 30 s and was nearly equivalent to other pauses before addition of G to the growing
transcript on these templates (Fig. 4E, note that pause RNA
is present in the first 3 lanes because many RNA polymerases
do not reach the pause site for up to 90 s under these
conditions). We conclude that there is a precise requirement
for the spacing between the pause RNA hairpin and the site
at which pausing occurs.
DISCUSSION
Transcription pausing by RNA polymerase was detected
first during transcription of the early portion of the luc operon
D ?RL455
S5pMGTP.Nus.4
pP
.
FIG. 4. Autoradiograms
from
gels containing
pause
RNAs
from
transcription
of the pRL455,
pRL459,
and pRL491
templates.
Synchronized
transcription
reactions
at 20 FM GTP without
NusA protein
or 2.5 PM GTP with 50 nM NusA protein
at 37 “C were performed
as described
under “Experimental
Procedures.”
Samples
were removed
from the reactions
at the times indicated
above the lane designations
and below. The
position
of the pause (I’) RNA is indicated
on the left. A, pRL455
(wild-type
trpL to position
+35 after the pause
site) template
at 20 pM GTP without
NusA. Lane I, sample removed
after 15 s; lane 2, sample removed
after 30 s;
lane 3, sample removed
after 45 s; lane 4, sample removed
after 1 min; lane 5, sample removed
after 1.5 min; lane
6, sample
removed
after 2 min; lane 7, sample
removed
after 2.5 min; lane 8, sample
removed
after 3 min. B,
pRL459
(position
of pause shifted one nucleotide
downstream)
template
at 20 pM GTP without
NusA. Samples
in
each lane are as for A. C, pRL491
(specifies
8 uridine
residues
immediately
after the pause site) template
at 20 PM
GTP without
NusA. Samples
in each lane are as for A. D, pRL455
(wild-type
trpL to position
+35 after the pause
site) template
at 2.5 pM GTP with 50 nM NusA.
Lane 1, sample removed after 30 s; lane 2, sample removed after
1 min; lane 3, sample removed
after 1.5 min; lane 4, sample removed
after 2 min; lane 5, sample removed
after 3
min; lane 6, sample removed
after 5 min; lane 7, sample removed
after 7 min; lane 8, sample removed
after 10 min.
E, pRL459
(position
of pause shifted
one nucleotide
downstream)
template
at 2.5 pM GTP with 50 nM NusA.
Samples
in each lane are as for D except that no sample taken after 10 min is present.
F, pRL491
(specifies
8
uridine residues
immediately
after the pause site) template
at 2.5 pM GTP with 50 nM NusA. Samples in each lane
are as for D.
Downstream
DNA
Sequences
in studies by Gilbert and Maizels (1, 3234). The presence of
GC-rich sequences one helical turn upstream from the pause
sites led them to suggest that difficulty in dissociating a stable
RNA:DNA
heteroduplex
could impede the progress of the
transcription
complex. Several other examples of so-called
“sequence-dependent”
pausing have been described (X 6 S
RNA, Refs. 2 and 35; E. coli rrnB leader, Ref. 36; phage T7
early region, Refs. 4, 33, 37; and the SV40 DNA F1 region,
Ref. 3). The mechanism
underlying
these pause events has
remained
obscure (5, 33). The most commonly
entertained
view has been that certain sequences, presumably
GC-rich,
downstream
from the catalytic site may be more difficult to
unwind and thus slow the transcription
complex (5, 32, 33).
In contrast, transcription
pausing associated with formation of an RNA hairpin
in the nascent transcript
(RNA
hairpin-induced
pausing) is a well-documented
component of
many bacterial attenuation
mechanisms
(7; Ref. 38 and references therein). In the best studied example of RNA hairpininduced pausing, which occurs in the leader region of the E.
coli trp operon, it has been shown that pausing (i) is sensitive
to the concentration
of the next nucleotide (GTP) to be added
after the pause (31, 39); (ii) is enhanced by the NusA protein
(40, 41); (iii) is reduced by base changes that destabilize the
RNA hairpin (6), but not by base changes in the loop region
of the pause RNA hairpin (6)“; (iv) can be enhanced or reduced
by amino acid substitutions
in the /3 subunit of RNA polymerase that similarly increase or decrease transcription
termination (42); (v) is detectable during transcription
in uiuo (43);
and (vi) is relieved by a ribosome translating
the trp leader
peptide coding region (44). Thus, the role of the RNA hairpin
as the causative agent of transcription
pausing has been
generally accepted.
Transcription
Pausing at the trpL Pause Site Is Influenced
by Downstream
DNA Sequences-We
have shown here that
the not-yet-transcribed
DNA sequence immediately
downstream from the site of pausing is an important
determinant
of pausing at the trpL pause site. Thus, the distinction
between RNA hairpin-induced
and sequence-dependent
pausing
is not clear-cut. Rather, both elements may contribute
to the
half-life of transcription
complexes paused during transcription of the trp leader region. Although
a significant
body of
evidence points to a role of RNA secondary structure
in
transcription
pausing (6, 7), a critical re-examination
of this
view may be in order, in light of the findings reported here.
Telesnitsky
and Chamberlin
(45) have described a similar
effect of downstream
sequence on the efficiency of termination at the phage T7Te terminator.
They found that changing
an AT-rich sequence to a GC-rich sequence immediately
past
the site that specifies the 3’ end of the terminated
RNA
reduces the efficiency of termination
at T7Te from 65 to 3%.
Thus the effect of downstream
sequences on elongation
by
RNA polymerase is not limited to transcription
pausing but
apparently
affects properties of the transcription
complex in
ways that influence both pausing and termination.
Our examination
of the effects on pausing of various DNA
sequences downstream
from the trpL pause site yielded two
other important
findings.
First, a run of uridine residues
positioned immediately
after the trpL pause site did not cause
transcription
termination
(Fig. 4). Thus, a p-independent
termination
site is not simply an RNA hairpin-induced
pause
site followed by a run of Us. As noted under “Results,”
we
have yet to test the effect of moving the uridine tract closer
to the center of dyad symmetry before the pause site; such a
construction
may cause termination.
Second, the spacing
between the position of RNA hairpin formation
and the site
of pausing is crucial. When the position of pausing at limiting
Modulate
Transcription
Pausing
15151
GTP was shifted one nucleotide downstream by repositioning
the critical G residue on the pRL459 and pRL520 templates,
pausing was nearly abolished (Table I, Fig. 4). This might be
due either to a spacing requirement
for location of the RNA
hairpin
in a binding
site on RNA polymerase
or to the
extension
by one nucleotide
of the segment of putative
RNA:DNA
hybrid or RNA-RNA
polymerase interaction
that
would not be disrupted by RNA hairpin formation.
Contacts between RNA Polymerase
and Double-stranded
DNA May Determine
the Effect of Downstream
DNA Sequences on Transcription
Pausing-How
might downstream
DNA sequences influence
the elongation
behavior of RNA
polymerase?
Shi et al. (46) have shown that E. coli RNA
polymerase
is able to transcribe
a template DNA strand to
within one nucleotide
of a psoralen cross-link between the
two strands of DNA. Since this cross-link
prevents melting
of the helix, a simple interpretation
of their data is that the
active site of RNA polymerase is located one base upstream
from the position
of DNA unwinding.
Such a topography
would require that, in a paused transcription
complex, the
DNA sequences downstream
from the pause site be doublestranded. However, the data of Shi et al. (46) do not rule out
unequivocally
the possibility
that the distance between catalytic and DNA-helix
unwinding
sites on RNA polymerase is
not fixed and that downstream
DNA sequences may, on
occasion, be single-stranded
(44). If so, many additional
effects of these sequences on the stability and catalytic efficacy
of the transcription
complex become possible. However, in
the absence of data requiring
consideration
of such complications, it is simplest to assume that the downstream
sequences we have examined are double-stranded
at the time of
transcription
pausing.
At least two explanations
are possible for how doublestranded DNA sequences downstream
from the catalytic site
might contribute
to the half-life of the paused transcription
complex. First, since this region of the DNA helix must be
unwound in order for the transcription
complex to translocate
forward, an exceptionally
high free energy requirement
to
break the hydrogen-bonded
base pairs in these sequences
could slow the rate of elongation. If so, one would predict that
GC-rich sequences would be more difficult to melt than ATrich sequences. However, we did not observe enhanced pausing when GC-rich sequences were placed downstream
from
the pause site (Table I). Indeed, an oligo(dC)
tract in the
nontranscribed
strand created as poor a pause site as
oligo(dT)
or oligo(dA) tracts and oligo(dG) and oligo(dA-dT)
tracts equivalently
cause the greatest reduction
in paused
transcription
complex half-life
(Table I). Thus, the strength
of base pairing in sequences downstream
from the pause site
does not explain the effect of these sequences on pausing.
The second possibility
is that contacts between RNA polymerase and the DNA helix downstream
from the catalytic
site influence the translocation
of the enzyme. Since difficulty
in unwinding
the downstream
DNA sequences appears to be
an unlikely
explanation,
such a model for the effects of
downstream sequence on pausing seems most tenable. Unfortunately, there is nothing distinguishing
about the wild-type
DNA sequence downstream
from the trpL pause site that
suggests a simple hypothesis.
We have considered
the possibility
that bending of the
downstream
DNA sequences is important.
Such a view has
been suggested to explain the effects of DNA sequences on
transcription
termination
by purified calf thymus RNA polymerase II (47). Here a correlation
between bending, caused
by two runs of dT nucleotides (dA nucleotides in the template
strand) phased one helical turn apart, and transcription
ter-
15152
Downstream
DNA Sequences Modulate
Transcription
Pawing
mination has been established (47). However, there are no
obvious phased dA or dT tracts in the sequence that follows
the trpL pause site. Moreover, replacement of an A with C at
+6, in the middle of the only dA tract within the downstream
region, had no significant effect on transcription pausing.
Telesnitsky and Chamberlin (45) have suggested that increased breathing of sequences downstream from the T7Te
termination site might facilitate chain release. Although we
have not explicitly examined breathing of the DNA downstream from the trpL pause site, it seems unlikely that reduced
pausing caused by both GC-rich and AT-rich sequences
(Table I) could be explained by increased or decreased breathing relative to the wild type. The downstream DNA sequences
that maximize chain release at a terminator might be significantly different from those that maximize transcription pausing. Elucidation of this relationship might yield clues to the
mechanisms of these two types of transcription elongation
control.
Thus we are left without a clear explanation for how the
DNA sequence downstream from the trpL pause site enhances
transcription pausing. Examination of a much larger number
of sequence variants would be required before a statistical
analysis of the effect of sequence composition on pausing
would be valid. At present, the simplest picture is offered by
consideration of the proposed three-dimensional structure of
RNA polymerase from Kornberg’s group (48). From electrondiffraction studies of the enzyme, they have suggested that
RNA polymerase contains a large cleft through which the
DNA template passes, much as for the known structure of the
Klenow fragment of E. coli DNA polymerase I (49). In this
cleft, RNA polymerase must interact with the template molecule. In principle, this interaction might be either through
specific contacts between amino acid sides chains and bases
such as are found in DNA-repressor complexes (50, 51), or
through a less specific electrostatic interaction. We favor the
latter view. Perhaps the overall shape of the helix determines
a charge distribution that may either facilitate or slow the
forward progress of RNA polymerase.
It is likely that similar interactions between RNA polymerase and the DNA template, RNA transcript, or RNA:DNA
heteroduplex occur on the upstream side of the transcription
complex (5, 11) and could influence the half-life of transcription pausing and the efficiency of transcription termination.
Demonstration of such a contribution, however, is complicated by the effect of sequence changes on the stability on
nascent transcript secondary structures. Examination of the
effects of various DNA sequences on pausing at a site completely devoid of potential secondary structures might circumvent this problem.
many other possible DNA sequences in this position significantly weaken the transcription pause (Table I). Thus, it
appears that both the RNA hairpin and the downstream DNA
sequence have evolved to form a strong transcription pause
site precisely where it would be required to synchronize transcription of the attenuator with translation of the leader
peptide control codons. This finding further strengthens the
view that transcription pausing is an integral component of
the attenuation mechanism.
The Effect of Downstream Sequences Suggests Transcription
Pausing at the trpL Pause Site Is a Component of the trp
Attenuation
Mechanism-Although
pausing by RNA polymerase is well documented, and can be detected in uiuo (43), as
22. Vieira,
23. Studier,
yet it has been impossible to prove that pausing is required
for proper regulation of transcription attenuation in amino
acid-biosynthetic operons (7, 52). A strong alternative has
been that the pause RNA hairpin serves the entirely distinct
function of “protector” against antiterminator (preemptor)
formation during folding of the leader transcript (53), and
that pausing by RNA polymerase is an accidental consequence
of the presence of the protector RNA hairpin. One counterargument to this view is that very similar pause sites are
found in the leader regions of different amino acid-biosynthetic operons, suggesting that pausing is a conserved function
(7). We show here that the DNA sequence after the trpL
pause site strongly favors transcription pausing and that
Acknowledgments-We
for useful discussions
thank
and helpful
C. Chan,
criticisms
R. Keene, and L. London
of the manuscript.
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