J. Mol. Biol. (2010) 396, 949–966
doi:10.1016/j.jmb.2009.12.025
Available online at www.sciencedirect.com
Motif III in Superfamily 2 “Helicases” Helps Convert the
Binding Energy of ATP into a High-Affinity RNA Binding
Site in the Yeast DEAD-Box Protein Ded1
Josette Banroques 1,2,3 , Monique Doère 3 , Marc Dreyfus 1 ,
Patrick Linder 3 and N. Kyle Tanner 1,3 ⁎
1
Institut de Biologie
Physico-chimique, CNRS UPR
9073 in association with the
Université Paris VII, Paris
75005, France
2
Centre de Génétique
Moléculaire, CNRS FRE 3144,
Gif-sur-Yvette 91198, France
3
Département de Microbiologie
et Médecine Moléculaire, Centre
Médical Universitaire, Geneva
1211, Switzerland
Received 6 August 2009;
received in revised form
8 December 2009;
accepted 14 December 2009
Available online
21 December 2009
Motif III in the putative helicases of superfamily 2 is highly conserved in
both its sequence and its structural context. It typically consists of the
sequence alcohol–alanine–alcohol (S/T-A-S/T). Historically, it was thought
to link ATPase activity with a “helicase” strand displacement activity that
disrupts RNA or DNA duplexes. DEAD-box proteins constitute the largest
family of superfamily 2; they are RNA-dependent ATPases and ATPdependent RNA binding proteins that, in some cases, are able to disrupt
short RNA duplexes. We made mutations of motif III (S-A-T) in the yeast
DEAD-box protein Ded1 and analyzed in vivo phenotypes and in vitro
properties. Moreover, we made a tertiary model of Ded1 based on the
solved structure of Vasa. We used Ded1 because it has relatively high
ATPase and RNA binding activities; it is able to displace moderately stable
duplexes at a large excess of substrate. We find that the alanine and the
threonine in the second and third positions of motif III are more important
than the serine, but that mutations of all three residues have strong
phenotypes. We purified the wild-type and various mutants expressed in
Escherichia coli. We found that motif III mutations affect the RNA-dependent
hydrolysis of ATP (kcat), but not the affinity for ATP (Km). Moreover,
mutations alter and reduce the affinity for single-stranded RNA and
subsequently reduce the ability to disrupt duplexes. We obtained intragenic
suppressors of the S-A-C mutant that compensate for the mutation by
enhancing the affinity for ATP and RNA. We conclude that motif III and the
binding energy of γ-PO4 of ATP are used to coordinate motifs I, II, and VI
and the two RecA-like domains to create a high-affinity single-stranded
RNA binding site. It also may help activate the β,γ-phosphoanhydride
bond of ATP.
© 2009 Elsevier Ltd. All rights reserved.
Edited by A. Pyle
Keywords: RNA helicase; ATPase; molecular motor; Saccharomyces cerevisiae;
RecA like
Introduction
*Corresponding author. IBPC, CNRS UPR Pierre et Marie
Curie, 75005 Paris, France. E-mail address:
[email protected].
Abbreviations used: SF2, superfamily 2; SF1,
superfamily 1; ssRNA, single-stranded RNA; AMP-PNP,
adenosine 5′-(β,γ-imino)triphosphate; PDB, Protein Data
Bank; SD-Leu plate, synthetic minimal medium plate
lacking leucine; 5-FOA, 5-fluoroorotic acid; EMSA,
electrophoretic mobility shift assay.
The putative helicases of superfamily 2 (SF2) are an
ubiquitous group of enzymes that are associated
with all processes involving RNA and DNA. SF2
proteins are closely related to those of superfamily 1
(SF1), some of which are known processive DNA
helicases that are involved in DNA replication and
repair.1,2 These NTPases (generally ATPases) are
characterized by a highly conserved structural core
consisting of two linked RecA-like domains that
contain seven or more conserved motifs involved in
nucleotide triphosphate and nucleic acid binding,
0022-2836/$ - see front matter © 2009 Elsevier Ltd. All rights reserved.
Motif III in Superfamily 2 “Helicases”
950
interdomain interactions, and NTPase activity.1,2
The largest SF2 family comprises DEAD-box
proteins, which are RNA-dependent ATPases and
ATP-dependent RNA binding proteins. These
proteins are associated with all processes involving
RNA from transcription to decay, and each family
member is typically involved in a unique
process.3–7 They contain nine conserved motifs
(Q, I, Ia, Ib, and II–VI), a conserved GG sequence
between motifs Ia and Ib, and a conserved QxxR
sequence (where x is any residue) between motifs
IV and V.3,4,7,8 Although commonly known as
DEAD-box “helicases,” they have not been shown
to have a processive unwinding activity of doublestranded RNA, and they are only able to displace
short duplexes, typically at very high protein/
duplex ratios in vitro.1,7,9
There are now a large number of solved crystal
structures of DEAD-box proteins, including several
with bound ligands.10–15 These data have verified or
clarified the roles of the different conserved motifs
and features. Thus, the Q motif is involved in
adenine recognition. Motifs I and II, also called
Walker motifs A and B, are involved in the binding
of the α and β phosphates of the ATP. Motifs Ia, Ib,
IV, and V; the conserved sequences GG and QxxR;
and a residue next to motif II are involved in singlestranded RNA (ssRNA) binding. Motifs III, V, and
VI are involved in binding the α, β, and γ
phosphates of ATP.2,4,7,8
The enzymatic roles of specific residues within the
motifs of DEAD-box proteins were derived from
mutagenic and biochemical analyses. The highly
conserved glutamic acid of motif II, which is
conserved in other SF2 and SF1 families as well, is
thought to activate the β,γ-phosphoanhydride bond
of ATP through a coordinated water molecule, in
association with a conserved histidine residue of
motif VI and a bound magnesium ion.4,16 A similar
mechanism of activation was proposed for the SF2
Dengue NS3 protein.17 The role of motif III is more
complicated; it has been suggested to link ATPase
and “helicase” activities because mutations in
mammalian eIF4A eliminated strand displacement
activity without affecting ATP hydrolysis or RNA
binding.18 In contrast, similar mutations in yeast
Has1 have reduced affinities for ATP and RNA and
reduced ATPase activity, which resulted in reduced
strand displacement activity.5 Mutations of motif III
also dissociated the ATPase activity from the strand
displacement activity in the related yeast DEAH-box
protein Prp22, but the RNA binding affinities were
not determined.19 Finally, motif III was proposed to
serve as a relay for ATP binding and hydrolysis and
for the binding of DNA in the SF1 Rep protein, but
the character of motif III and its interactions with the
other motifs in SF1 are significantly different from
those of SF2.20,21
We were interested in obtaining mutants of motif
III in the DEAD-box protein Ded1 that dissociated
the different activities, so that we could better
understand the interrelationships between the
motifs and the role of the protein in vivo. DED1 is
an essential gene in the yeast Saccharomyces cerevisiae. It was first identified as an intragenic suppressor of a PRP8 mutant, which implied a role in
mRNA splicing,22 but it was also implicated in the
transcription of polymerase III RNAs,23 and to have
a general role in translation initiation24,25 and 40S
ribosome scanning.26 Finally, it was implicated in Pbody formation and RNA degradation27 and in
yeast L-A virus synthesis.28 Ded1 is closely related
to a subfamily of DEAD-box proteins involved in
developmental regulation,29–31 including the Drosophila Vasa protein for which the crystal structure
was solved in the presence of RNA and adenosine
5′-(β,γ-imino)triphosphate (AMP-PNP).12
Motif III is defined by the sequence serine–
alanine–threonine (S-A-T) in the DEAD-box family.
We mutated the three positions in DED1 and
analyzed the in vivo phenotypes and in vitro
properties of the proteins expressed in and
purified from Escherichia coli. We find that mutations of motif III are poorly tolerated in vivo, and
that these mutants affect the hydrolysis of ATP
(kcat) and the affinity for ssRNA, but not the
affinity for ATP (Km). This subsequently reduces
the ability of the proteins to displace nucleic acid
duplexes. It appears that motif III is critical for
aligning—and perhaps activating—the γ phosphate of ATP, for coordinating the different motifs,
and for helping to convert the binding energy of
ATP into a high-affinity RNA binding site. Thus,
mutations of motif III in Ded1 affect the observed
“helicase” activity indirectly by altering and
reducing RNA binding, and this may be true for
other characterized SF2 proteins as well.
Results
Sequence alignments in and around motif III
Sequence alignments of 699 unique DEAD-box
proteins were performed as described in Materials
and Methods and are shown in Fig. 1a. Motif III
contained a serine in the first position 89% of the
time, while a threonine occurred 11% of the time.
The second position of alanine was 100% conserved,
and there was virtually always a threonine in the
last position, although a serine was found in 0.4% of
the sequences. In contrast, the sequences flanking
motif III were more variable, but there was a
tendency for hydrophobic residues to precede and,
to a lesser extent, follow motif III. We examined the
solved crystal structures of Vasa, DDX19B (human
Dbp5), Mss116, and eIF4AIII in the presence of
bound RNA and AMP-PNP to better understand its
structural context;10–15 motif III occurred at the end
of a β-sheet strand that subsequently turned into a
loop, and this was probably why there was a
preference for hydrophobic groups preceding the
motif (Fig. 1a). The loop structure then turned into a
helix, but there were differences between Vasa,
DDX19B, Mss116, and the different eIF4AIII crystal
structures in the size of the loop and helix.
Motif III in Superfamily 2 “Helicases”
951
Fig. 1. Sequence alignments
show that motif III in SF2 proteins
is highly conserved. (a) Nearly
700 unique DEAD-box protein
sequences were aligned as previously described.32 The sequence
shown below is from Ded1. The
structural context of motif III is
based on the solved crystal structure of Vasa, where L is a loop, S
is a β-sheet strand, and H is a
helix. Alanine of motif III interacts
with γ-PO4 of AMP-PNP through
a water molecule in the Vasa
structure. (b) Sequence alignments
of various families of SF2 proteins.
Upper-case letters are residues,
and lower-case letters are classes
of residues: a, aromatic; c,
charged; h, hydrophobic; l, aliphatic; o, serine or threonine; p,
polar; t, alanine or glycine; (−)
negative; (+) positive; (•) any
residue. The consensus was arbitrarily taken at 60% of the aligned
sequences.
We examined other solved crystal structures of
DEAD-box proteins in the Protein Data Bank
(PDB)†. We found the structural context of motif
III to be remarkably conserved, with the first residue
being almost always part of a β-sheet, followed by a
loop of four (4.3 ± 0.7) and then a helix. The latter
helix was followed by a loop, a β-sheet strand, and
then a loop that connected domain 1 with domain 2.
Although there were differences in loop size in the
presence and in the absence of ligands, as for Mss116
and eIF4AIII, these differences were not correlated
with the ligands per se. Instead, they seemed to
reflect the variability between the various solved
crystal structures.
We did BLAST and sequence alignments on other
SF2 families as well and found motif III to be highly
conserved between families (Fig. 1b). For example,
motif III was serine–alanine–threonine (S-A-T) in
† http://www.rcsb.org/
over 99% of the DEAH-box proteins. The other
families, such as Ski2 and Hef, showed more
variability, but the consensus was always alcohol–
alanine–alcohol, with threonine being largely preferred in the third position. The SecA and Snf2
families were exceptions; the consensus was threonine–glycine–threonine in nearly 100% of the
sequences (Fig. 1b). Despite this remarkable conservation, the sequences flanking motif III were highly
variable between families. Nevertheless, the structural context found in the DEAD-box family was
also true for the solved crystal structures of all the
other SF2 families examined in the PDB. Some
families, such as SecA and Hef, had longer loops
(generally six to seven), and the viral NS3 proteins
had loops that connected directly with domain 2.
But the context of motif III (β-sheet loop) was almost
inevitably the same. However, the alcohol– tiny–
alcohol sequence of motif III was not universally
conserved in SF2 families or even in the DEAD-box
family; the UL9 protein of the herpes simplex virus
952
had a D-A-T sequence,33 and the cyanobacteria cold
shock DEAD-box protein CrhC, which was not
obtained in the original BLAST, had the sequence
F-A-T. 34 These results suggested that the first
position of motif III was somewhat malleable in SF2.
Growth phenotypes of motif III mutants
We randomly mutagenized the first codon
(corresponding to S341) and the third codon (T343)
of motif III in DED1, transformed yeast cells with the
DNA, selected the transformed cells on synthetic
minimal medium plates lacking leucine (SD-Leu
plates), and then streaked the resulting colonies on
5-fluoroorotic acid (5-FOA) plates to eliminate cells
retaining the plasmid-encoded copy of the wild-type
DBP1 gene, which complemented DED1 deletion
when overexpressed. We used yeast cells deleted for
the chromosomal copies of both DED1 and DBP1
genes in all our screens. About 270 colonies were
tested in each case on 5-FOA plates at 18 °C, 30 °C,
and 36 °C. For position S341, we found that 50% of
the clones were wild type or pseudo wild type,
about 20% of the clones failed to grow at any
temperature, and about 30% showed conditional
growth (Fig. 2a). The plasmid DNA was recovered
from representative colonies, retransformed into
Motif III in Superfamily 2 “Helicases”
yeast, replated on SD-Leu plates, and restreaked
on 5-FOA plates to verify the phenotypes. We
sequenced 10 of the recovered wild-type and
pseudo-wild-type plasmids, 13 of the conditional
mutants, and 5 plasmids from cells that did not
grow on 5-FOA plates. For position T343, we found
that about 35% of the clones were wild type or
pseudo wild type, 60% failed to support growth,
and only 5% were unable to grow at low or high
temperatures (Fig. 2a). We sequenced 13 of the
recovered wild-type or pseudo-wild-type plasmids,
6 of the conditional mutants, and 10 plasmids of the
lethal mutants.
Figure 2b summarizes the results obtained in these
screens. Consistent with sequence alignments, we
noticed that the DED1 mutants that supported
growth to about the same level as wild type
contained either a threonine or a serine in the first
or third position of the motif, respectively, although
S341T grew slightly slower than the wild type at all
the temperatures tested and T343S was slightly cold
sensitive. However, the first position tolerated a
number of other amino acids that allowed the cells
to grow more or less than the wild type. Two
categories of conditional mutants were seen according to the strength of the phenotypes. The mutants
S341A, S341G, and S341C had weak slow-growth
Fig. 2. Motif III mutations have
strong phenotypes. (a) Cultures of
yeast cells, deleted for chromosomal copies of DED1 and DBP1
(ded1∷HIS dbp1∷KANMX6) and
transformed with leucine plasmids
containing ded1 with the indicated
mutations, were serially diluted (by
factors of 10) and then spotted on
minimal medium plates containing
5-FOA to eliminate cells retaining
the uracil plasmid containing the
wild-type copy of DBP1. Plates
were incubated at the indicated
temperatures. (Ø) A control leucine
plasmid lacking any insert. Note
that the few isolated colonies seen
for some lanes (SAC at 18 °C and
SAI) were pseudo revertants that
resulted from recombination between plasmids. (b) Résumé of the
growth phenotypes of the tested
mutants. CS, cold sensitive; TS,
sensitive to high temperatures.
Motif III in Superfamily 2 “Helicases”
phenotypes and were considered pseudo wild type.
In contrast, the S341H, S341L, S341M, and S341Q
mutants were more severe, and they were unable to
grow at 18 °C and grew more slowly at 30 °C and
36 °C. Three mutants (S341R, S341D, and S341P)
were unable to grow at any temperature. Thus,
bulky and charged groups were less tolerated than
small residues in the first position.
We only obtained the conditional mutation T343C
for the third position for all six clones isolated,
indicating an essential role for an alcohol at this
position. Nevertheless, the T343C mutant was
severe, and it failed to support growth at 18 °C
(Fig. 2a). None of the other mutants were viable
(T343A, T343I, T343F, T343Y, T343M, T343E, T343K,
and T343N). We also site-specifically mutated the
100% conserved A342 into a glycine; this mutant
grew very poorly at 30 °C or 36 °C and was unable
to grow at 18 °C. Finally, we constructed a double
mutant where both S341 and T343 were changed to
alanines (S341A T343A). This mutant failed to
support growth at all temperatures.
This study was not intended to be an exhaustive
screen; we were mostly interested in obtaining
conditional mutations for subsequent analyses. However, different codons were obtained for the same
residues, indicating that the oligonucleotides were
sufficiently randomized. None of the mutants showed
dominant-negative phenotypes in the presence of the
wild-type DED1. Protein expression of the mutants
was confirmed by Western blot analysis (data not
shown). We concluded from these in vivo analyses
that the third position of motif III was more important
than the first, but that an alcohol residue (S, T, and, to
a lesser extent, C) was preferred for both positions.
We were interested in knowing how general these
phenotypes were in other essential yeast DEAD-box
proteins. We previously showed that, in Has1, the
first-position S228A mutant was cold sensitive, and
the third position T230A was lethal.5 In this study, we
953
mutated S201 and T203 in eIF4A to alanine and
obtained the same results: S201A was cold sensitive,
while the T203A mutant failed to support growth
(data not shown). In contrast, when we independently
mutated T188 and T190 in Dbp8 to alanines, we
obtained wild-type growth at all the temperatures
tested, although the combined double mutant grows
two times slower at 30 °C (data not shown).35
However, Dbp8 was more tolerant of mutations in
general, relative to other essential DEAD-box proteins,
and its ATP-dependent enzymatic activity per se may
not have been essential in vivo (J.B., unpublished data).
Isolation of intragenic suppressors of the
conditional T343C Ded1 mutant
We decided to investigate gene-encoded (intragenic) changes that compensate for mutations in
motif III and that might reveal something about how
other residues potentially interact with motif III or
about its role in the activities of Ded1. We chose the
ded1 T343C mutant because cells transformed with
this plasmid were unable to grow at 18 °C or lower
temperatures. We performed a PCR mutagenesis of
the plasmid-encoded gene containing the SAC
mutation as described in Materials and Methods.
We obtained 12 clones out of the 250 screened clones
that grew better at the selected temperatures. We
obtained the intragenic suppressors S118P and
E143G twice; F205L, F242L, K301R, Y359H, Y359N,
and M421V once; and two plasmids that contained
two mutations in addition to T343C (L152S + T370A
and I360T + S590P). We separated these latter mutations by subcloning them into the appropriate
regions of the original ded1-T343C plasmid; only
T370A and I360T enhanced the growth of the motif
III mutant, while L152S and S590P did not affect
growth. Thus, we obtained 10 unique intragenic
mutations that suppressed the T343C mutation to
various extents (Fig. 3).
Fig. 3. Intragenic suppressors of
SAC mutation. Cells containing the
wild-type gene (SAT), T343C mutation (SAC), and T343C mutation
with intragenic suppressors (+ residue) were plated as described in
Fig. 2.
954
The M421V, E143G, S118P, F205L, and F245L
suppressors only slightly enhanced growth at 30 °C
and 36 °C, and poorly suppressed the cold-sensitive
phenotype. K301R moderately enhanced growth at
all temperatures. Oddly, T370A moderately suppressed the cold-sensitive phenotype, but enhanced
the high-temperature sensitivity. The best suppressors were I360T, Y359H, and Y359N, suggesting that
this region of the protein was particularly important.
Interestingly, the Y359H suppressor grew better at
all temperatures, while Y359N grew best at higher
temperatures.
We mapped the positions of the suppressors on
the three-dimensional model of Ded1 (Fig. 4b). All
suppressors, except for S118P, were located on the
outer shell of the two RecA-like domains and were
outside the conserved motifs. Nevertheless, nearly
all the suppressors mapped to residues that had
functionally conserved side chains among the 700
aligned DEAD-box sequences. The S118P suppressor was located in the highly variable amino
terminus in a region that was predicted to be
unstructured. The E143G mutant was adjacent to
the highly conserved, isolated, aromatic group of
the DEAD-box Q motif;36,37 this position was
Motif III in Superfamily 2 “Helicases”
typically polar (83.7%) in sequence alignments,
but a glycine naturally occurred 10.0% of the time.
F205L was found in a variable loop region
between motifs I and Ia. F242L was adjacent to
motif Ia; it was a leucine in only 4.4% of the
sequences, while a polar group was present in
56.7%. K301R was close to motif II, and it was an
arginine in 11.3% of the sequences, charged in
67.7% and polar in 95.3%. The position occupied
by Y359 was typically hydrophobic (92.3%) and
often a proline (69.2%); it was an asparagine in
2.0% of the sequences and never a histidine.
Similarly, I360T was a hydrophobic residue in
75.8% of the sequences and a threonine in only 3%.
T370A was located close to a variable loop, but
was typically polar (57.1%). Finally, the position
corresponding to M421V was polar (65.4%), and a
valine was present in only 0.4% of the sequences.
None of the suppressors were close to residues
predicted to interact with the RNA ligand, and
only S118P was positioned close enough to
potentially affect the interactions with AMP-PNP.
However, the E143G mutation could alter the
conserved helix–loop–helix–loop structure of the Q
motif involved in adenine recognition.
Fig. 4. Three-dimensional modeling of Ded1. The tertiary structure of Ded1 was modeled based on the solved crystal
structure of chain A of Vasa as described in Supplementary Material. (a) The structure of Vasa (orange) superimposed on
the ribbon model of Ded1 (gray with colored motifs). Vasa is shown with 40% transparency to facilitate viewing of the two
structures. The conserved motifs are as indicated, and the bound ligands (AMP-PNP and RNA) are from the Vasa
structure and are shown as stick models. The bound Mg2+ ligand is shown as a space-filling model. (b) Positions of the
suppressors of the motif III T343C mutation (boxed) on the model of Ded1. The structure is shown from the back of that
shown in (a), and the ribbons outside the conserved motifs are shown with 40% transparency for viewing the mutant
residues (sticks). (c) Alignment of the Vasa and Ded1 proteins that was generated with the Swiss-Model program (see
Supplementary Material). The conserved motifs and sequences are indicated above the alignment, and the positions of the
T343C mutation and corresponding suppressors are shown below (boxed). Motifs are colored as shown in (a) and (b).
Motif III in Superfamily 2 “Helicases”
955
Reduced RNA-dependent ATPase activities of
motif III mutants
We subcloned representative ded1 mutants into a
pET expression vector containing a carboxyl-terminal His6 tag. This allowed us to express the proteins
in E. coli and to purify them on nickel affinity
columns. We were able to successfully obtain the
wild-type Ded1, various motif III mutants, and the
SAC mutant with six of the different suppressors at
high purity and yield (data not shown). Moreover,
we purified the Y359H and Y359N suppressors
separated from the original SAC mutation. Finally,
we purified the mutants Y359K and Y359P—the
former to determine whether an amine-containing
residue was important at this position and the latter
because a proline residue was found in this position
nearly 70% of the time in sequence alignments. The
expression and purification characteristics of the
proteins were similar to those of wild type,
indicating that there were no major structural
alterations. However, we verified this by subjecting
the mutant proteins SAC, SAA, and AAA to partial
trypsin digestion in the presence and in the absence
of ligands; there were no significant differences in
cleavage patterns with those of the wild-type
protein (data not shown).
We then tested the mutants and the suppressors
for their capacity to hydrolyze ATP in the
presence of RNA by using a molybdate–Malachite
Green assay to monitor the production of free
phosphate. We used total yeast RNA, as previously described, because the in vivo substrate for
Ded1 is unknown.32,36,37 We then determined the
Michaelis–Menten kinetic parameters at 30 °C
(Table 1).
A very low enzymatic performance (kcat/Km) in
vitro, relative to the wild type, was observed for the
lethal (T343A and T343I) and the poorly growing
(T343C and A342G) ded1 mutants at the second and
third positions. They all had between 5.6% and 8.0%
of wild-type activity. This result confirmed the
importance of these two positions in motif III.
Similar changes from the first-position S341 to
cysteine and alanine had less profound effects (24–
55% wild-type activity), which again was well
correlated with the in vivo phenotypes. These
mutants grew nearly as well as the wild type, and
the percentage of activity corresponded to the
severity of the growth defect. Oddly, the S341L
mutant had better ATPase activity than the T343C
mutant (12% versus 6.8%), but it grew more slowly
than T343C at higher temperatures (both were cold
sensitive); the leucine mutation may have destabilized the protein. Thus, the first position, which
showed more sequence variability in the alignments,
was less critical than the second and third positions,
which were more highly conserved. The lethal
double mutant ded1 S341A T343A showed only
2.9% of the ATPase activity of the wild type with
1 mM ATP, and we were unable to determine its
enzymatic performance (data not shown). Interestingly, the effects were almost entirely on the rate of
hydrolysis of the ATP (kcat) rather than on the
binding affinities (Km), which showed only small
differences for all the mutants.
Table 1. Kinetic parameters for RNA-dependent ATPase activities
Wild type (SAT)
AAT
CAT
LAT
SGT
SAA
SAC
SAI
SAS
AAA
SAC + F242L
SAC + K301R
SAC + Y359H
SAC + Y359N
SAC + I360T
SAC + T370A
Y359H
Y359N
Y359K
Y359P
Km (mM)
kcat (min− 1)a
kcat/Km × 10− 3 (M− 1 min− 1)b
% Wild typec
Phenotypesd
EMSAe
0.37 ± 0.03
0.23 ± 0.03
0.44 ± 0.06
0.53 ± 0.10
0.35 ± 0.02
0.52 ± 0.05
0.48 ± 0.05
0.57 ± 0.04
0.58 ± 0.04
ND
0.40 ± 0.08
0.21 ± 0.02
0.093 ± 0.013
0.23 ± 0.03
0.26 ± 0.03
0.095 ± 0.006
0.063 ± 0.002
0.18 ± 0.01
0.20 ± 0.01
0.18 ± 0.02
280 ± 30
94 ± 9
79 ± 8
46 ± 5
21 ± 2
22 ± 2
24 ± 2
30 ± 4
110 ± 10
ND
31 ± 3
25 ± 3
31 ± 3
26 ± 3
35 ± 4
26 ± 3
150 ± 20
150 ± 20
190 ± 20
200 ± 20
740 ± 70
410 ± 60
180 ± 30
87 ± 17
60 ± 6
41 ± 4
51 ± 5
51 ± 6
190 ± 20
ND
78 ± 16
120 ± 10
330 ± 50
110 ± 20
140 ± 10
270 ± 30
2400 ± 200
820 ± 90
960 ± 100
1100 ± 100
100
55
24
12
8.0
5.6
6.8
6.9
26
∼3
10
16
44
15
18
37
320
110
130
150
WT
∼ WT
∼ WT
CS and SG
bSG
NG
CS and SG
NG
∼ WT
NG
CS and ∼SG
CS and ∼SG
∼ WT
CS
CS
∼CS and TS
WT
WT
WT
WT
+++
++
ND
+/−
+/−
+/−
+
+/−
+++
+/−
++
++
++
++
++
++
+++ ½
+++ ½
ND
ND
Values were derived from nonlinear fits to the Michaelis–Menten equation using the mean values of at least three independent
experiments. Errors are standard deviations from the mean.
a
A minimum of 10% error was assumed for the protein concentrations.
b
The largest errors of kcat or Km were carried forward.
c
Enzymatic performance (kcat/Km) relative to the wild-type protein. AAA value was estimated using 1 mM ATP.
d
WT, wild-type growth; ∼WT, pseudo-wild-type growth; SG, slow growth; ∼ SG, slightly slow growth; bSG, very slow growth; NG,
no growth; CS, cold sensitive; ∼ CS slightly cold sensitive; TS, sensitive to high temperatures.
e
AMP-PNP-dependent EMSAs of RNA. Note that the SAA, SAI, SGT, and AAA mutants did not form discrete retarded products.
EMSA and strand displacement activities were correlated.
956
The suppressors of the T343C mutation improved
enzymatic performance by 47% to nearly 650%,
although the best suppressor was only 44% of the
wild-type activity. Oddly, this was almost entirely
due to an increased affinity (lower Km) for ATP.
Moreover, the best suppressor Y359H showed
nearly the same properties in isolation of the
T343C mutation: it had nearly 6-fold higher affinity
for ATP relative to that for the wild type, while kcat
was reduced by only 2-fold. The Y359K, Y359N, and
Y359P mutants, in isolation, improved Km by about
2-fold and had similar values of kcat as Y359H. Thus,
motif III mutations affected the hydrolysis of ATP
rather than the binding, and the suppressors
compensated for reduced hydrolysis by enhancing
the affinity for ATP.
We used highly saturating concentrations of
total yeast RNA (0.5 μg/μl) for RNA-dependent
ATPase assays, based on the wild-type Ded1.
However, it was possible that the mutants had a
very poor affinity for the RNA and that our assay
was not saturating for these proteins. We were not
able to use more than 1.5 μg/μl total RNA in our
assays because the components tended to precipitate under the reaction conditions. Thus, it was
possible that the reduced rates of hydrolysis (kcat)
were indirectly a result of a reduced affinity for the
RNA.
Motif III in Superfamily 2 “Helicases”
Motif III mutants had reduced ATP-dependent
affinity for RNA
We then investigated the RNA binding affinities of
the proteins by electrophoretic mobility shift assays
(EMSAs).38,39 We previously showed that Ded1 has a
higher affinity for ssRNA substrates in the presence of
AMP-PNP, which is a nonhydrolyzable analog of
ATP, than in the absence of a nucleotide or in the
presence of ADP.32 In this set of experiments, we used
a constant concentration of a 25-nt-long ssRNA
(RNA01) and varied the protein concentration as
described in Materials and Methods.
In the absence of nucleotide or in the presence of
ADP, all of the motif III mutants showed weak
binding to ssRNA, similar to binding to the wild-type
protein, although the AAA mutant was particularly
weak (Fig. 5). All of the motif III mutants showed
strongly reduced affinities for ssRNA in the presence
of AMP-PNP that was correlated with the in vivo
phenotypes and in vitro ATPase activities. The SGT
mutant was often retained in the well of the gel (25–
50% of retarded material), suggesting that it might
aggregate or that it was less stable (data not shown).
To facilitate these analyses, we typically counted
everything that migrated above the free ssRNA as
being retarded. Therefore, our estimates actually
overestimated the binding affinities because smearing
Fig. 5. EMSAs of RNA incubated with different Ded1 constructs. 32P-end-labeled RNA01 was incubated with different
concentrations (nM) of the indicated Ded1 proteins in the absence of a cofactor (−nt), in the presence of ADP (+ ADP), or in
the presence of AMP-PNP (+ PNP), and electrophoretically separated on a 6% nondenaturing polyacrylamide gel at 4 °C.
Free, RNA01 migration in the absence of protein; Ori, the bottom of the loading well of the gel. Bar graphs show the
percentage of radioactivity migrating more slowly than the free RNA01. Note that the SAT + Y359H panel was chosen
because it best showed the three distinct slower-migrating bands; typically, it had slightly stronger binding affinity than
the wild type, as determined in Hill plots with more extensive data points (data not shown).
Motif III in Superfamily 2 “Helicases”
above the free ssRNA was counted similarly as
discrete bands migrating slowly in the polyacrylamide gel. Blunt-ended double-stranded RNA and
single-stranded DNAs showed a weak affinity for the
wild-type protein, independent of AMP-PNP.32
As shown in Fig. 5, motif III mutations affected
both the affinity and the character of ssRNA binding
in the presence of AMP-PNP. Typically, the wildtype Ded1 showed three distinct bands that were
clearly separated from the free ssRNA and that
increased with the protein concentration. In contrast, the most benign motif III mutation (AAT)
showed only one major band that smeared upward
at the higher protein concentrations and that
corresponded to the faster-migrating band of the
wild-type Ded1. The other mutants showed binding
characteristics that corresponded to the phenotypes.
For example, the SAA and SAI mutants, which did
not support growth, did not form a discrete band,
while the SAC mutant, which was slow growing
and cold sensitive, formed a single weak band
(Fig. 5). The AAA mutant showed very little AMPPNP-dependent binding; it did not support growth.
The suppressors Y359H and T370A (Fig. 5) and the
suppressors F242L, K301R, Y359N, and I360T (data
not shown) enhanced the RNA binding affinity of
the SAC mutant in direct relation to their in vivo
phenotypes and in vitro ATPase activities (kcat/Km),
but they did not restore the characteristic profile of
the binding (a single major band instead of three
bands). In contrast, the Y359H suppressor in
isolation showed essentially wild-type binding
properties with three major bands in the presence
of ssRNA and AMP-PNP (Fig. 5).
We determined the binding affinities of wild-type
Ded1, Y359H, and Y359N in the presence and in the
absence of AMP-PNP using the Hill plot, as
previously described (data not shown).32 There
was significant day-to-day experimental variability,
and the differences were small, but the isolated
suppressors consistently showed a higher affinity
for the ssRNA than for the wild type in the presence
and in the absence of AMP-PNP. The medium value
was about a 30% enhancement. There was no
evidence that the wild-type or isolated suppressor
proteins were functioning cooperatively as multimers or that there were multiple independent
binding sites. Thus, these results were consistent
with the interpretation for the Y359 suppressors that
the enhanced affinity for ATP was indirectly a result
of a slightly enhanced affinity for ssRNA, although
cumulative effects could not be ruled out.
Reduced strand displacement activity of motif III
mutants
We analyzed the various purified proteins by a
strand displacement assay, as described in Materials
and Methods. We used different substrates that
formed duplexes on either the 5′ end or the 3′ end of
the ssRNA “landing” site for the proteins, including
a modified version of K01 (K06) that gave less
protein-independent displacement because it dis-
957
rupted a hairpin structure that formed around the
hybridization site. Substrates had 25-nt-long ssRNA
landing sites and 16-bp duplexes. Both 5′ and 3′
duplexes were functional substrates. We used a 20fold or 25-fold excess of a DNA trap (α-Hyb1 DNA)
that hybridized to the 16-nt-long 32P-labeled Hyb1
oligonucleotide that was released and prevented it
from reannealing onto the ssRNA template (Fig. 6a).
However, these experiments were qualitatively and
quantitatively similar to those obtained when an
excess of unlabeled Hyb1 (which hybridized to form
an identical but unlabeled duplex) was used.32 In
both cases, the single-stranded regions of the
unlabeled RNA products were competitors for the
binding site on the protein that increased during the
course of the reactions. Thus, the observed displacement rates decreased during the course of the
reactions.
We tested both a DNA version and an RNA
version of Hyb1 (Fig. 6a); the DNA–RNA duplex was
nearly completely displaced after 5 min at 30 °C with
a 50-fold excess of substrate to wild-type Ded1. In
contrast, the RNA–RNA duplex, which was calculated to have a Gibbs free energy that was 5.5 kcal/
mol more stable (ΔG° = −25.3 kcal/mol) than that of
the DNA–RNA duplex (ΔG° = − 19.8 kcal/mol)
under standard conditions, was only partially displaced after 60 min with stoichiometric concentrations of the duplex and the protein (Fig. 6b; wild-type
Hyb1 RNA/K06 RNA). However, this more than
100-fold difference in activities was not entirely
attributable to the difference in the free energies of
the duplexes because the DNA trap formed an
unusually weak duplex with the released Hyb1
RNA, which was calculated to be 8.3 kcal/mol less
stable than the RNA–RNA duplex due to weak rUU/
dAA base pairs (ΔG° = −17.0 kcal/mol).40 Indeed,
25% of the [32P]Hyb1 RNA/K06 RNA duplex would
reform in the presence of a hundredfold excess of αHyb1 DNA when heat denatured and slow cooled in
the absence of protein, while only about 5% of [32P]
Hyb1 DNA/K06 RNA duplex would reform with
only a 25-fold excess of α-Hyb1 DNA (data not
shown). Hence, we primarily used the DNA/RNA
duplexes in our subsequent analyses.
We then assayed our various motif III mutants
and suppressors (Fig. 6b). We used a GKT-to-GAT
mutant of the P-loop of motif I as control because
this mutant showed only insignificant ATPase
activity, and it was expected to significantly disrupt
the α and β phosphate interactions of ATP with the
protein; this mutant showed slight ATP-independent strand displacement at a 10-fold excess over the
DNA–RNA substrate. The various motif III mutants
showed intermediate activities that were strongly
correlated with their ATPase (kcat/Km) and RNA
binding activities. Thus, the double AAA mutant
showed weak strand displacement at a 10-fold
excess of protein over duplex, while the SAC mutant
showed a relatively good strand displacement with
a 5-fold excess of the duplex over protein. The
suppressors enhanced the strand displacement of
the SAC mutation according to their ATPase and
958
Motif III in Superfamily 2 “Helicases”
Fig. 6. Strand displacement activity of the various Ded1 mutants. (a) Three of the duplexes used in these assays.
P-labeled Hyb1 DNA or RNA was hybridized to R1 or K06 RNAs as shown. α-Hyb1 DNA is complementary to
Hyb1, and it acts as a trap to prevent rehybridization of [32P]Hyb1 on the RNAs. (b) Strand displacement activity of
50 nM duplex by the various Ded1 constructs, in the presence (1 mM) or in the absence of ATP. Samples were
incubated for the indicated times (in minutes) at 30 °C, and then the material was electrophoretically separated and
analyzed as described in Materials and Methods. Note that the protein concentrations were calibrated to give similar
amounts of displacement, when possible, with the same amount of duplex; therefore, they reflect relative differences
in activity between the wild-type protein and the mutants. The duplexes used for each panel are as indicated. (‖) The
displaced [32P]Hyb1 trapped by α-Hyb1.
32
RNA binding activities, with SAC + Y359H being
about the same as the wild type (Fig. 6b and data not
shown). However, there appeared to be no correlation with the characteristic binding profile (formation of three discrete bands) of ssRNA binding, as
seen with the wild-type protein. Moreover, the in
vivo phenotypes were more severe than expected
from the strand displacement activities, relative to
previous mutants in the Q motif and motif IV.32,36
The AAA mutant, which did not support growth,
had approximately the same ATP-dependent strand
displacement activity as the wild-type yeast eIF4A
with the same substrate.41 The SAA mutant enzymatically displaced a 5-fold excess of duplex even
though it contained a lethal mutation. This suggested that this assay imperfectly reflected the in
vivo enzymology of Ded1 with respect to motif III.
Structural context of motif III
The structural context of motif III was highly
conserved in all the solved SF2 crystal structures
examined; it was situated at the end of a β-sheet
strand that goes into a loop. For most SF2 proteins,
motif III was an integral part of domain 1; however,
in the viral NS3 proteins, it was part of a flexible
linker that went directly into domain 2.21 Figure 7b
shows motif III interactions in the solved crystal
structure of Vasa with the bound ligands, but
virtually identical interactions were seen in the
solved crystal structures of DDX19B and Mss116
and in the two solved structures of eIF4AIII in the
presence of bound ligands (PDB coordinates 2db3,
2hyi, 3i5x, 3i5y, 3i61, 3i62, 2j0s, 3fht, and 3g0h).10–15
Serine 432 of motif III in Vasa (S341 in Ded1)
formed hydrogen bonds with D402 of motif II and
with the peptide backbone of T434 of motif III. The
peptide backbone of S432 interacted with the
backbone of A287 that was two residues upstream
of motif I, although this interaction was not seen in
one of the crystal structures of eIF4AIII (PDB
coordinate 2j0s), DDX19B (PDB coordinate 3g0h),
and Mss116 (PDB coordinate 3i5x); the residues
were slightly outside the limits of detection for a
hydrogen bond (N3.3 Å). The peptide backbone of
the central A433 of motif III hydrogen bonded
with γ-PO4 of AMP-PNP through a water molecule, and this interaction was shared with E400 of
motif II that is thought to activate γ-PO4 in
association with H575 of motif VI.4,16 Threonine
434 of motif III formed hydrogen bonds with D402
of motif II and with H575 of motif VI; this latter
interaction was not seen in one of the DDX19B
structures (PDB coordinate 3fht), but the threonine
and the histidine were only 2.90 Å apart. Thus,
motif III was centrally located to bring together
elements of motifs I, II, and VI, as well as to help
position γ-PO4 of ATP.
Motif III in Superfamily 2 “Helicases”
Fig. 7. Interactions of motif III in the solved crystal
structure of Vasa. (a) Overview of the relationship between
the conserved motifs and the substrates. The conserved
motifs are shown as ribbons, and the bound ligands (AMPPNP and oligouridine U2–U6) are shown as sticks. Domains
1 and 2 are shown as half-ellipses, and the overall
relationship is similar to the structure shown in Fig. 4a. (b)
Interactions of motif III. Only interactions involving motif III
are shown, and the equivalent residues in Ded1 are shown
in parentheses. The two water molecules are shown as small
red dots for clarity, important residues are shown as sticks,
and hydrogen-bond interactions are shown as dotted green
lines. H2Oc has been proposed to catalyze the hydrolysis of
ATP, while H2Or was proposed to enhance the nucleophilicity of H2Oc.14,42,43 The bound Mg2+ ligand is shown as a
gray sphere. Glutamine 400 (E307) of motif II (not shown)
makes contacts with γ-PO4 of AMP-PNP through one of the
same water molecules (H2Or) as A433 (A342) of motif III.
The only SF2 crystal structure solved in various
reaction states with the various bound ligands was
that of the Dengue virus NS3 protein.17 This protein
forms nearly identical interactions with the RNA
959
and AMP-PNP ligands as Vasa and eIF4AIII,17
which are similar to those of DDX19B.13,15 Mss116
forms more extensive interactions with the RNA,
both within the core and with the carboxyl
terminus.14 Moreover, the NS3 proteins show the
same characteristic cation–π interactions as the
DEAD-box proteins between a conserved arginine
of motif VI and a conserved phenylalanine of motif
IV.32 Thus, even though the NS3 motif III was part
of the flexible linker between domains, it showed the
same types of interactions described above for Vasa
in nearly all the ligand states, including the
hydrolyzed form of ADP and PO4. This was true
even though the equivalent residues in motifs II and
VI were histidine and glutamine, respectively,
instead of aspartic acid and histidine, and motif III
had the sequence TAT. ATP was hydrolyzed when
diffused into RNA-NS3 crystals, which indicated
that the crystals were catalytically active. The
interactions in NS3 seemed to be independent of
the presence of nucleotide; most of the structural
differences are RNA dependent in other regions of
the protein.17 The sole exception was the structure
bound to RNA and free PO4, which showed no
interaction between the alanine of motif III and PO4
through water. These types of interactions are also
true for Mss116 in the presence of ADP and BeFx or
AlFx (PDB coordinates 3i61 and 3i62)14 and for
eIF4AIII in the presence of ADP and AlFx (PDB
coordinate 3ex7). 42 BeFx is isomorphous to a
phosphate group; thus, ADP-BeFx is considered a
ground-state ATP analog, while AlFx is thought to
mimic the planar phosphate transition state.44
We concluded that the motif III interactions were
highly conserved in DEAD-box proteins and in
many other SF2 families as well, even if the identity
of specific residues varied between families. Indeed,
when we independently changed the aspartic acid
of motif II to a histidine and the histidine of motif VI
into a glutamine in Ded1, such as in DEAH-box and
NS3 proteins, we obtained nearly wild-type growth;
however, the interactions also were genetically
linked because combining either mutation with the
SAC mutation of motif III gave a lethal phenotype (J.
B., unpublished data). Thus, although the residues
could vary, the nature of the interactions of motif III
remained the same in many different SF2 proteins.
Discussion
Motif III was previously thought to link ATPase
activity with a “helicase” activity in SF2
proteins.5,18,19,45 In our hands, it appears that the
role of motif III in the DEAD-box protein Ded1 is
more complex than this. The mutants affect the
hydrolysis of ATP (kcat) and the binding affinity and
binding characteristics for ssRNA; this subsequently
reduces the observed strand displacement activity.
There are few effects on the binding affinity for ATP
(Km), even though the central alanine of motif III
makes contact with γ-PO4 of ATP through water in
the solved crystal structures. The observed in vitro
960
activities correlate well with in vivo phenotypes,
which indicate that they also are important in the cell.
However, the mutant phenotypes that we obtained
are more severe than those obtained in other motifs of
Ded1 that we have studied. For example, the mutant
F405Y in motif IV of Ded1 has more than 10-fold less
ATPase activity but nearly wild-type growth,32 while
the LAT mutation of motif III has 8-fold less activity
but barely grows. This emphasizes the central role
that motif III plays in the in vivo activity of Ded1. This
effect seems to be primarily at the level of the
characteristics of RNA binding. All the mutants that
failed to form discrete bands in the presence of AMPPNP either did not support growth (SAA, SAI, and
AAA) or supported barely detectable growth (SGT;
Fig. 1 and Table 1). In contrast, the strand displacement activity in vitro was more closely correlated
with the overall AMP-PNP-dependent binding affinity for the RNA (smearing); for example, the SAA
mutant is able to enzymatically displace a 5-fold
excess of duplex (as opposed to at least a 50-fold
excess by the wild type) in the presence of ATP even
though it is not viable in vivo (Figs. 5 and 6).
Role of the intragenic suppressors of
SAC mutation
The suppressors mapped almost entirely on the
outer shell of the “helicase” core throughout the two
RecA-like domains (Fig. 4b and c). The only
exception was the S118P suppressor that mapped
26 residues upstream of the isolated highly conserved phenylalanine of the Q motif in a region that
is likely to be flexible.36,37 The E143G suppressor
mapped next to this phenylalanine, which is further
evidence for the critical, but as yet unknown, role of
the phenylalanine in the enzymatic activity of
DEAD-box proteins. Nearly all the suppressors
mapped in positions with functionally conserved
amino acids (in nearly 700 aligned sequences), and
they often involved changing a hydrophobic residue
into a polar or charged group, or vice versa. All the
analyzed suppressors compensate for the reduced
catalytic efficiency of the SAC mutant by enhancing
the affinity for ATP (Km) rather than by restoring the
rates of hydrolysis (kcat; Table 1). They could do this
directly by modifying the ATP binding site or
indirectly by enhancing the binding affinity for
ssRNA. The latter is at least partially the case for the
Y359H and Y359N suppressors because both confer
an enhanced affinity for ssRNA in the presence and
in the absence of AMP-PNP. This is consistent with
the cooperative ATP-dependent RNA binding that
we observe.
However, none of the suppressors map close to
the RNA binding site identified in the solved crystal
structures of Vasa, DDX19B, Mss116, and eIF4AIII,
and only S118P is in a flexible loop that is close to the
adenine.10–15 They are all located on the exterior
solvent-exposed shell of the core, so it is unlikely
that they indirectly affect the ligand binding sites
(ATP or ssRNA) by altering the conformations of the
RecA-like domains. Moreover, the in vivo pheno-
Motif III in Superfamily 2 “Helicases”
types and in vitro properties are closely correlated,
so it also is unlikely that the suppressors primarily
affect interactions with other cofactors. The suppressors alter the predicted electrostatic potential of
the protein surface of Ded1, and this could enhance
protein–protein or protein–RNA contacts (data not
shown). However, we have no functional (enzymatic) evidence that Ded1 dimerizes or oligomerizes,
and the subunits of any such oligomer would have
to work independently. Moreover, no dimerization
is observed in proteome screens in vivo.46,47 On the
other hand, the suppressors Y359H, Y359N, I360T,
and K301R could enhance RNA binding by introducing polar or positively charged residues, but this
presupposes a much larger RNA binding site than is
currently known. This nevertheless remains a real
possibility for Ded1 because they could expand the
preexisting site or create new interactions that
facilitate ssRNA binding. This would explain why
they enhance the affinity for ssRNA without
restoring the wild-type binding profile (Fig. 6b).
Previous characterization of motif III in other
SF2 proteins
There is a notable divergence of observations for
the in vivo and in vitro effects of the mutations of
motif III on different SF2 proteins. An SAT-to-LAT
mutation in the related DDX3 in humans has
significantly reduced ATPase and strand displacement activities, as seen for Ded1.48 Changing SAT to
AAA in the mammalian DEAD-box protein eIF4A
increased the binding affinity (lower Km) for ATP,
increased Vmax, but eliminated the strand displacement activity; RNA binding is unchanged, but this
was based indirectly on the level of RNA-stimulated
UV cross-linking of α-[32P]ATP to the protein.18 The
equivalent yeast eIF4A protein has a cold-sensitive
phenotype for the AAT mutation and a lethal
phenotype for SAA (this study). Likewise, the
mutations AAT and SAA in the yeast DEAD-box
protein Has1 have cold-sensitive or lethal phenotypes, respectively; 2-fold or 4-fold reduced affinities
for ssRNA, respectively; 2-fold reduced affinities for
ATP (higher Km); 2-fold lower rates of hydrolysis
(kcat); and reduced strand displacement activities.5
In the yeast Dbp8 protein, the mutations AAT and
SAA are pseudo wild type (this study), and the AAA
mutant undergoes slow growth at 30 °C.35
A lethal phenotype is obtained when SAT is
changed to LAT in the yeast DEAD-box protein
Sub2.49 The mitochondria yeast DEAD-box protein
Mss116 likewise has reduced ATPase and strand
displacement activity when SAT is changed to
AAA.50,51 The RNA binding affinity is similar to
that for the wild type, but Mss116 has an expanded
RNA binding site that may have reduced the
magnitude of the mutant effects.14,51 Similar results
are seen with YxiN, which has a secondary highaffinity binding site.52 Mutants of the yeast Prp28
protein, where the central alanine is changed to
valine or tryptophan, are wild type or cold sensitive,
respectively;53 however, the ATPase activity of
Motif III in Superfamily 2 “Helicases”
Prp28 may not have been essential under the assay
conditions.32 This also may explain why more than
half of the proteins having a LAT mutation in a large
screen of DEAD-box, DEAH-box, and Ski2 proteins
involved in ribosomal processing in yeast were wild
type: the endogenous wild-type genes were under
the control of tetracycline-repressible promoters that
may have been leaky enough to partially compensate for the mutations (J.B., unpublished data).54,55
This would clarify the weak phenotypes of some of
the mutations in the other conserved motifs as well.
The in vivo phenotypes of mutations of serine or
threonine in the SAT motif of the SF2 DEAH-box
proteins Prp2, Prp16, Prp22, and Prp43 in yeast are
similarly either slow growing or cold sensitive.19,56–58
Mutations of either threonine in the TAT sequence of
viral NPH-II eliminate the strand displacement
activity and significantly reduce the ATPase
activity.45 Likewise, similar mutants of the NS3
protein of hepatitis C virus have reduced ATPase
and strand displacement activities.59,60 Finally, a
threonine-to-serine mutation in the DAT sequence
of the UL9 protein of herpes simplex virus has
reduced replication and reduced ATPase activity,
without affecting the affinity for ATP.33,61 Changing
the alanine to serine also reduces replication, while a
threonine-to-alanine mutation destroys replication
competence.33
Even though there are significant differences in
observations and interpretations, there appears to be
a common theme for the mutations of motif III that
is found in the various SF2 proteins. Alterations in
either alcohol often result in cold-sensitive slowgrowing or lethal phenotypes, and the thirdposition alcohol is more critical than the firstposition alcohol. Mutations affect ATPase and
strand displacement activities, and they have less
profound effects on the binding affinity of ATP.
Mutations may or may not affect the overall binding
affinities for nucleic acids (RNA or DNA). However,
most of these results are compatible with our
observations on Ded1, and the apparent differences
probably reflect the characteristics of specific proteins. For example, some proteins, such as the
DEAD-box protein YxiN, have an additional highaffinity RNA binding site that is separable from the
RecA-like core and therefore independent of ATP
binding.52,62 Some proteins, such as Mss116, have
more expansive RNA binding sites that may hide
the effects of altered binding around the enzymatic
site in vitro.14 In other cases, the ATPase activity per
se may not be necessary for in vivo viability.
What is the role of motif III?
Motif III is defined by the sequence alcohol–tiny–
alcohol. The question remains: Why are the characteristic features of motif III so highly conserved in SF2
proteins? The role of the conserved alanine seems
clear: it forms hydrogen bonds with γ-PO4 of ATP
through a bound water molecule that is also shared
with the highly conserved glutamic acid of motif II;
this water molecule has been proposed to act as part
961
of a proton relay system that enhances the nucleophilicity of the attacking water molecule.14,42,43 The
glutamic acid is thought to activate the β,γ-phosphoanhydride bond with a water molecule, in
association with the conserved histidine of motif VI
in DEAD-box proteins (or glutamine in other
families) and a bound Mg2+ .4,16,17 The alanine
seems to help position the functional groups, but it
may also be involved in activating the β,γ-phosphoanhydride bond. This explains why only alanine
—and, to a lesser extent, glycine—is used because a
larger side group would cause steric hindrance with
γ-PO4; with residues in motifs I, II, and VI; and with
the bound water molecule.
The role of serine or threonine in the first position
in SF2 helicases also seems clear. Serine, threonine,
and cysteine are the only residues that can form
hydrogen bonds with the peptide backbone of the
third residue and maintain the sharp bend of motif
III seen in SF2 proteins (but not in SF1). The first
residue further positions motif III relative to motif II
by making contacts with the second aspartic acid of
motif II in DEAD-box proteins (or a histidine in
other families). There also are peptide backbone
interactions between the alcohol and an amino acid
just upstream of motif I that could help position
motif I relative to motifs II and III. However, the
side-chain interactions of the first-position serine are
not critical because neutral residues (A and G) are
tolerated under our assay conditions.
The third-position threonine links motifs II, III,
and VI in SF2 proteins through the interactions of
the motif II aspartic acid (histidine in other families)
and the motif VI histidine (glutamine). This threonine plays a critical role in enzymatic activity
because only mutants with an alcohol at this
position are viable in Ded1. Nevertheless, it is not
clear why threonine is preferred at this position in
nature because serine can form the same interactions. Moreover, the SAS mutant supports nearly
wild-type growth and has good enzymatic activities
under our experimental conditions. Hydroxyl alcohols at positions 1 and 3 are probably preferred
because they are weakly acidic, are good nucleophiles, and readily form hydrogen bonds. In
contrast, we expect proteins substituted with
cysteines to form weaker hydrogen bonds and to
be more sensitive to pH.
DEAD-box proteins are thought to use ATP
hydrolysis to recycle enzymatic forms rather than
to drive the ssRNA binding or strand displacement
activities.7,63,64 We have previously shown that
Ded1 binds ATP and ADP with approximately the
same affinities (Km and Ki, respectively), and we
were able to isolate mutants that altered the affinity
for one without altering the affinity for the other.36
In yeast eIF4A, we showed that ADP actually binds
more tightly than ATP even though there are no γPO4 interactions.37 Therefore, the binding energy of
the β and γ phosphates is used not only to activate
the phosphoanhydride bond but also to constrain
the different motifs and the two RecA-like domains
of the protein core, so that they can bind RNA with a
962
much higher affinity. This explains why mutations
of motif III in mammalian eIF4A actually enhance
the binding affinity for ATP because the other
elements are no longer constrained.18 RNA binding
would further activate the β,γ-phosphoanhydride
bond for the hydrolysis reaction to occur by an
activated water molecule, presumably through
distortion of the bond or stabilization of a transition
state. In accordance with this idea, an AAA
mutation in YxiN reduces the cooperative binding
and coupling energies of ATP and RNA, even
though the overall relationship between domains 1
and 2 (“closed” and “open” forms) is not different
from that of the wild-type protein in the presence
and in the absence of ligands.52 In Förster resonance
energy transfer analyses, YxiN maintains the
“closed” conformation in association with RNA
and with AMP-PNP, ADP-BeFx, ADP-AlFx, and
ADP-MgFx, although the latter two analogs do not
support RNA unwinding, indicating that there are
subtle conformational differences.65
This would be consistent with the proposed
mechanisms on how catalytic antibodies and
enzymes use ligand binding energies to stimulate
reactions by stabilizing the transition states.66,67
ATP binding to the protein would create a highaffinity RNA binding site, but RNA binding to the
protein would subsequently activate the β,γ-phosphoanhydride bond for hydrolysis. The resulting
ADP-bound form would then have a low affinity for
the RNA, and translocation or release of the RNA
would occur. Motif III is ideally situated to
coordinate the ATP and the different motifs for
these conformational changes. Thus, previously
published claims that motif III links the ATPase
and “unwinding” activities need to be reconsidered
to reflect the possibility that the unwinding activity
was indirectly effected by an altered or reduced
affinity for the nucleic acid ligand within the
enzymatic site, or to reflect the possibility that
RNA binding and strand displacement are concurrent, and that the ATPase activity is only used to
recycle the protein from a high-affinity conformation to a low-affinity conformation for ligands.
Materials and Methods
Sequence alignments and phylogeny
BLAST and sequence alignments with ClustalXXL were
performed as previously described.32 The Bork Web site
was used to determine the consensus sequence‡, and the
ExPASy Stataln tool§ was used to quantify the results. We
examined the structural context of motif III in the solved
crystal structures of various SF2 families using the
following PDB coordinates: 1cu1, 1d9z, 1db3, 1fuu, 1hei,
1hv8, 1m74, 1nkt, 1nl3, 1oyw, 1oyy, 1q0u, 1qde, 1qva,
1s2m, 1t6n, 1tf2, 1tf5, 1vec, 1wp9, 1wrb, 1xti, 1xtj, 1xtk,
‡ http://coot.embl.de/Alignment/consensus.html
§ http://www.expasy.ch/tools/stataln.html
Motif III in Superfamily 2 “Helicases”
1yks, 1ymf, 1z3i, 1z63, 1z6a, 2bmf, 2db3, 2f55, 2fsf, 2fsg,
2fsh, 2fsi, 2fz4, 2g9n, 2gxu, 2hxy, 2hyi, 2i4i, 2j0q, 2j0s, 2oxc,
2p6r, 2p6u, 2pl3, 2v6i, 2v6j, 2va8, 2vbc, 2vlx, 2z0m, 3b7g,
3ber, 3bor, 3bxz, 3ews, 3fhc, 3fht, 3g0h, 3i5x, 3i5y, 3i61,
3i62, and 8ohm.
Cloning, vectors, and strains
All yeast manipulations were performed using standard
techniques.68 The DED1 coding region (1850 bp), cloned
into the SpeI and XhoI sites of Bluescript plasmid
(Stratagene), was used for all PCR amplifications and
clonings. Mutations were introduced by a two-step overlapping fusion PCR, using PFU polymerase as previously
described.36,37 Mutations were introduced with oligonucleotides completely randomized for the three positions of
the codon at either amino acid position 341 or amino acid
position 343. The two pools of amplified DNAs were
digested with AccI and StyI, gel purified, and cloned into
the equivalent sites of DED1 in the Bluescript plasmid.
Additional oligonucleotides were used to specifically
insert either alanine or cysteine at position 341, and a
glycine at position 342. A double mutant with alanine in
both positions 341 and 343 was also made. SpeI-XhoI
fragments corresponding to the entire coding region of
DED1 were then subcloned into the p415-PL-ADH vector
(ARS-CEN-LEU).37 Ded1 yeast plasmids were transformed
into the Δded1/Δdbp1 strain (ded1∷HIS3 dbp1∷KANMX6)
containing the DBP1 open reading frame cloned into the
multicopy p416-TEF plasmid;69 using the DBP1 plasmid
instead of DED1 minimized recombination between
plasmids and the appearance of pseudo revertants. For
each screen, we selected about 260–280 transformants that
could grow on SD-Leu plates, streaked them on a 5-FOAcontaining medium, and then tested them for their ability
to grow at 16 °C, 18 °C, 30 °C, and 36 °C. Selected
constructs were recovered by plasmid rescue, their
phenotypes were verified by retransforming yeast, and
then they were sequenced to identify the mutation. Protein
expression was verified by Western blot analysis, with
antibodies against the HA tag present in the constructs.
Isolation of intragenic suppressors
Intragenic suppressors of the conditional DED1 T343C
mutation were obtained by mutagenic PCR using Taq
DNA polymerase and two oligonucleotides that hybridized at either end of the ded1 T343C open reading frame.
Two to four independent cycles of mutagenic PCR were
first performed using 6.5 mM MgCl2, 0.5 mM MnCl2,
1 mM dCTP and dTTP, and 0.2 mM dATP and dGTP.
Then, 40 PCR cycles were performed under normal
conditions using 2% of the mutagenized PCR as
template. This library of DNA fragments was digested
with SpeI and XhoI and subcloned into the equivalent
sites of the p415-ADH vector. The DNA was amplified
in E. coli and then used to transform the Δded1/Δdbp1
yeast strain containing the DBP1 plasmid. We restreaked
250 clones on 5-FOA plates at 16 °C, 18 °C, 30 °C, and
36 °C to eliminate cells retaining the wild-type DBP1
plasmid. The plasmids that supported growth were
recovered and subjected to restriction digestion mapping. All the clones that showed wild-type growth were
eliminated because they were recombinants with the
Dbp1 plasmid, and we ended up with 12 clones that
grew better than the original mutant. We verified the
phenotypes of the plasmids by retransforming yeast, and
then we sequenced the DNA.
Motif III in Superfamily 2 “Helicases”
963
Protein expression and purification
Strand displacement assay
The AgeI fragments of DED1 containing the motif III
mutations were cloned into the equivalent sites of the pET22b expression vector (Novagen) containing the wild-type
DED1. The Rosetta(DE3) E. coli strain (Novagen) was used
for expressing the proteins. Expression and purification
were performed as previously described.32,36 Protein
concentrations were determined by the Bio-Rad Protein
Assay, using bovine serum albumin as standard. The
proteins were judged to be 90–95% pure on Laemmli SDS
polyacrylamide gels.
We used various duplex substrates, including K01–
Hyb1 and R1–Hyb1, that had been previously
described.32,36 We also used a new 45-nt-long substrate,
K06, that was similar to K01, except for the single-stranded
region had an additional uridine and had a cytidine
changed to a uridine (5′ GGG CUA GCA CCG UAA AGC
AAG UUA AUU CAA AAC AAA ACA AAA GCU 3′,
where underlined characters hybridized to the oligonucleotide and boldface characters were different from K01);
this was performed to destabilize a hairpin structure that
could compete for the Hyb1 binding site. This construct
gave less background displacement than the K01–Hyb1
duplex. The same 16-nt-long oligonucleotide (Hyb1)
hybridized to either the 5′ end (K01 and K06) or the 3′
end (R1) of the RNAs. We used both DNA and RNA
variants of Hyb1 (Hyb1 DNA and Hyb1 RNA) that were
labeled on the 5′ ends with 32P. The DNA–RNA duplexes
had a calculated Gibbs free energy of −19.8 kcal/mol,40,70
while the RNA–RNA duplexes had − 25.3 kcal/mol.71
However, the actual melting temperatures of the different
duplexes varied due to competing intramolecular basepairings of the RNAs.
Under our reaction conditions, with a large excess of
substrate over the enzymes, most of the displaced Hyb1
would reanneal on the RNAs. Consequently, we generally
used a 20-fold to 25-fold excess of an unlabeled 18-nt-long
oligonucleotide complementary to Hyb1 (α-Hyb1 DNA)
as trap to prevent reannealing of the displaced [32P]Hyb1.
The α-Hyb1 DNA–Hyb1 DNA duplex had a calculated
Gibbs free energy of − 18.4 kcal/mol, and the α-Hyb1
DNA–Hyb1 RNA duplex was − 17.0 kcal/mol; the lowerthan-expected free energy of the latter was due to the
unusually low stability of the two sets of rUU/dAA base
pairs.40
Reactions were carried out at 30 °C for various times,
the products were separated by 15% polyacrylamide gel
electrophoresis at 4 °C under nondenaturing conditions,
and the corresponding bands were quantified with a
Cyclone PhosphoImager and Optiquant software and
analyzed with Kaleidagraph. Because the proteins had a
low intrinsic affinity for the RNA substrates, we often saw
ATP-independent strand displacement that was directly
proportional to the protein concentration. It was low for
the Ded1 wild-type protein, which was used at a low
protein–duplex ratio, but we had significant background
for many of the poorly active mutants that were used at
much higher protein concentrations. Moreover, there was
some strand exchange with the oligonucleotide trap,
which resulted in an apparent protein-independent
displacement during the time course. This background
activity was subtracted from the results.
RNA-dependent ATPase assays
We used a colorimetric assay based on molybdate–
Malachite Green and used whole-yeast RNA (Type III
Sigma) and ATP (GE-Pharmacia) as substrates, as
previously described for Ded1.36 In brief, reactions
were incubated at 30 °C for various times and stopped
by making the reaction mix 4.5 mM in ethylenediaminetetraacetic acid and placing it on ice. Data were
analyzed using Kaleidagraph 4.0.4 (Synergy). Time
courses at each ATP concentration were usually
repeated three independent times. Time courses also
were carried out in the absence of RNA to determine
the background hydrolysis of the ATP and in the
absence of ATP to monitor contribution due to RNA
degradation. In both cases, the signals were only a
small percentage of the reactions including ATP and
RNA; these background signals were subtracted from
subsequent calculations of the reaction rates. The mean
values and standard deviations from the mean were
calculated with Kaleidagraph.
Measuring protein–RNA affinity by EMSA
We used a 5′ 32P-end-labeled 25-nt-long RNA that was
chemically synthesized (RNA01; PAGE purified; Dharmacon), as previously described.32 In brief, we used a
constant RNA concentration (5 nM) and varied the protein
concentration. The protein and RNA were incubated
together in 20-μl volumes for 20 min on ice with no
nucleotide, with 5 mM ADP (Sigma), or with 5 mM AMPPNP (Roche). RNasin (1 U/μl; Promega) was added to
reactions containing AMP-PNP because of RNase contamination in the product. Loading buffer was then
added, and the products were separated by electrophoresis on 6% nondenaturing polyacrylamide gels run at 4 °C.
Data were quantified with either a Cyclone (Packard) or
Typhoon Trio (Amersham Biosciences) PhosphoImager
and ImageQuant software (Packard), and then analyzed
with Kaleidagraph. Most mutants poorly bound to the
RNA, and binding values could not be determined. For
the wild type and for some suppressors in isolation of
other mutations, we used a more extensive range of
protein concentrations so that we could fit the data to the
Hill equation using a single binding site, as previously
described (although similar results were obtained when
the number of binding sites was left unfixed).32 To
facilitate comparisons, we counted all of the radioactivities
above the free oligonucleotide as protein-specific binding
even though there were significant qualitative differences.
Background smearing of radioactivity, from the lane
without added protein, was subtracted from the results.
All binding assays were generally conducted multiple
times for each protein.
Acknowledgements
We thank Ronald Bock, Tien-Hsien Chang, and
Paul Lasko for the kind gifts of plasmids. We are
very much indebted to Costa Georgopoulos for his
undying support for our endeavors. This work was
supported by the Swiss National Science Foundation, the Canton of Geneva, the Centre National de
la Recherche Scientifique, and ANR Program Blanc
grant 08-BLAN-0086-02 to M.D. N.K.T. was supported by the Swiss Institute of Bioinformatics and
964
as a CDD Chercheur of Centre National de la
Recherche Scientifique. J.B. also was supported by a
Short-Term European Molecular Biology Organization Fellowship.
Supplementary Data
Supplementary data associated with this article
can be found, in the online version, at doi:10.1016/
j.jmb.2009.12.025
References
1. Pyle, A. M. (2008). Translocation and unwinding
mechanisms of RNA and DNA helicases. Annu. Rev.
Biophys. 37, 317–336.
2. Singleton, M. R., Dillingham, M. S. & Wigley, D. B.
(2007). Structure and mechanism of helicases and
nucleic acid translocases. Annu. Rev. Biochem. 76, 23–50.
3. Bleichert, F. & Baserga, S. J. (2007). The long unwinding
road of RNA helicases. Mol. Cell, 27, 339–352.
4. Cordin, O., Banroques, J., Tanner, N. K. & Linder, P.
(2006). The DEAD-box protein family of RNA
helicases. Gene, 367, 17–37.
5. Rocak, S., Emery, B., Tanner, N. K. & Linder, P. (2005).
Characterization of the ATPase and unwinding
activities of the yeast DEAD-box protein Has1p and
the analysis of the roles of the conserved motifs.
Nucleic Acids Res. 33, 999–1009.
6. Silverman, E., Edwalds-Gilbert, G. & Lin, R. J. (2003).
DExD/H-box proteins and their partners: helping
RNA helicases unwind. Gene, 312, 1–16.
7. Tanner, N. K. & Linder, P. (2001). DExD/H box RNA
helicases: from generic motors to specific dissociation
functions. Mol. Cell, 8, 251–262.
8. Caruthers, J. M. & McKay, D. B. (2002). Helicase
structure and mechanism. Curr. Opin. Struct. Biol. 12,
123–133.
9. Jankowsky, E. & Fairman, M. E. (2007). RNA
helicases—one fold for many functions. Curr. Opin.
Struct. Biol. 17, 316–324.
10. Andersen, C. B., Ballut, L., Johansen, J. S., Chamieh,
H., Nielsen, K. H., Oliveira, C. L. et al. (2006). Structure
of the exon junction core complex with a trapped
DEAD-box ATPase bound to RNA. Science, 313,
1968–1972.
11. Bono, F., Ebert, J., Lorentzen, E. & Conti, E. (2006). The
crystal structure of the exon junction complex reveals
how it maintains a stable grip on mRNA. Cell, 126,
713–725.
12. Sengoku, T., Nureki, O., Nakamura, A., Kobayashi, S.
& Yokoyama, S. (2006). Structural basis for RNA
unwinding by the DEAD-box protein Drosophila Vasa.
Cell, 125, 287–300.
13. von Moeller, H., Basquin, C. & Conti, E. (2009). The
mRNA export protein DBP5 binds RNA and the
cytoplasmic nucleoporin NUP214 in a mutually
exclusive manner. Nat. Struct. Mol. Biol. 16, 247–254.
14. Del Campo, M. & Lambowitz, A. M. (2009). Structure
of the yeast DEAD box protein Mss116p reveals two
wedges that crimp RNA. Mol. Cell, 35, 598–609.
15. Collins, R., Karlberg, T., Lehtio, L., Schutz, P., van den
Berg, S., Dahlgren, L. G. et al. (2009). The DEXD/Hbox RNA helicase DDX19 is regulated by an α-helical
switch. J. Biol. Chem. 284, 10296–10300.
Motif III in Superfamily 2 “Helicases”
16. Story, R. M., Li, H. & Abelson, J. N. (2001). Crystal
structure of a DEAD box protein from the hyperthermophile Methanococcus jannaschii. Proc. Natl Acad. Sci.
USA, 98, 1465–1470.
17. Luo, D., Xu, T., Watson, R. P., Scherer-Becker, D.,
Sampath, A., Jahnke, W. et al. (2008). Insights into
RNA unwinding and ATP hydrolysis by the flavivirus
NS3 protein. EMBO J. 27, 3209–3219.
18. Pause, A. & Sonenberg, N. (1992). Mutational analysis
of a DEAD box RNA helicase: the mammalian
translation initiation factor eIF-4A. EMBO J. 11,
2643–2654.
19. Schwer, B. & Meszaros, T. (2000). RNA helicase
dynamics in pre-mRNA splicing. EMBO J. 19,
6582–6591.
20. Korolev, S., Hsieh, J., Gauss, G. H., Lohman, T. M. &
Waksman, G. (1997). Major domain swiveling
revealed by the crystal structures of complexes of E.
coli Rep helicase bound to single-stranded DNA and
ADP. Cell, 90, 635–647.
21. Korolev, S., Yao, N., Lohman, T. M., Weber, P. C. &
Waksman, G. (1998). Comparisons between the
structures of HCV and Rep helicases reveal structural
similarities between SF1 and SF2 super-families of
helicases. Protein Sci. 7, 605–610.
22. Jamieson, D. J., Rahe, B., Pringle, J. & Beggs, J. D.
(1991). A suppressor of a yeast splicing mutation
(prp8-1) encodes a putative ATP-dependent RNA
helicase. Nature, 349, 715–717.
23. Thuillier, V., Stettler, S., Sentenac, A., Thuriaux, P. &
Werner, M. (1995). A mutation in the C31 subunit of
Saccharomyces cerevisiae RNA polymerase III affects
transcription initiation. EMBO J. 14, 351–359.
24. Chuang, R. Y., Weaver, P. L., Liu, Z. & Chang, T. H.
(1997). Requirement of the DEAD-box protein ded1p
for messenger RNA translation. Science, 275,
1468–1471.
25. de la Cruz, J., Iost, I., Kressler, D. & Linder, P. (1997).
The p20 and Ded1 proteins have antagonistic roles in
eIF4E-dependent translation in Saccharomyces cerevisiae. Proc. Natl Acad. Sci. USA, 94, 5201–5206.
26. Berthelot, K., Muldoon, M., Rajkowitsch, L., Hughes,
J. & McCarthy, J. E. (2004). Dynamics and processivity
of 40S ribosome scanning on mRNA in yeast. Mol.
Microbiol. 51, 987–1001.
27. Beckham, C., Hilliker, A., Cziko, A. M., Noueiry, A.,
Ramaswami, M. & Parker, R. (2008). The DEAD-box
RNA helicase Ded1p affects and accumulates in
Saccharomyces cerevisiae P-bodies. Mol. Biol. Cell, 19,
984–993.
28. Chong, J. L., Chuang, R. Y., Tung, L. & Chang, T. H.
(2004). Ded1p, a conserved DExD/H-box translation
factor, can promote yeast L-A virus negative-strand
RNA synthesis in vitro. Nucleic Acids Res. 32,
2031–2038.
29. Johnstone, O., Deuring, R., Bock, R., Linder, P., Fuller,
M. T. & Lasko, P. (2005). Belle is a Drosophila DEADbox protein required for viability and in the germ line.
Dev. Biol. 277, 92–101.
30. Tarn, W. Y. & Chang, T. H. (2009). The current
understanding of Ded1p/DDX3 homologs from yeast
to human. RNA Biol. 6, 17–20.
31. Rosner, A. & Rinkevich, B. (2007). The DDX3
subfamily of the DEAD box helicases: divergent
roles as unveiled by studying different organisms
and in vitro assays. Curr. Med. Chem. 14, 2517–2525.
32. Banroques, J., Cordin, O., Doere, M., Linder, P. &
Tanner, N. K. (2008). A conserved phenylalanine of
motif IV in superfamily 2 helicases is required for
Motif III in Superfamily 2 “Helicases”
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
cooperative, ATP-dependent binding of RNA substrates in DEAD-box proteins. Mol. Cell. Biol. 28,
3359–3371.
Martinez, R., Shao, L. & Weller, S. K. (1992). The
conserved helicase motifs of the herpes simplex virus
type 1 origin-binding protein UL9 are important for
function. J. Virol. 66, 6735–6746.
Chamot, D., Magee, W. C., Yu, E. & Owttrim, G. W.
(1999). A cold shock-induced cyanobacterial RNA
helicase. J. Bacteriol. 181, 1728–1732.
Daugeron, M. C. & Linder, P. (2001). Characterization
and mutational analysis of yeast Dbp8p, a putative
RNA helicase involved in ribosome biogenesis.
Nucleic Acids Res. 29, 1144–1155.
Cordin, O., Tanner, N. K., Doere, M., Linder, P. &
Banroques, J. (2004). The newly discovered Q motif of
DEAD-box RNA helicases regulates RNA-binding
and helicase activity. EMBO J. 23, 2478–2487.
Tanner, N. K., Cordin, O., Banroques, J., Doere, M. &
Linder, P. (2003). The Q motif: a newly identified
motif in DEAD box helicases may regulate ATP
binding and hydrolysis. Mol. Cell, 11, 127–138.
Cann, J. R. (1998). Theoretical studies on the mobilityshift assay of protein–DNA complexes. Electrophoresis,
19, 127–141.
Lane, D., Prentki, P. & Chandler, M. (1992). Use of gel
retardation to analyze protein–nucleic acid interactions. Microbiol. Rev. 56, 509–528.
Sugimoto, N., Nakano, S., Katoh, M., Matsumura, A.,
Nakamuta, H., Ohmichi, T. et al. (1995). Thermodynamic parameters to predict stability of RNA/DNA
hybrid duplexes. Biochemistry, 34, 11211–11246.
Barhoumi, M., Tanner, N. K., Banroques, J., Linder, P.
& Guizani, I. (2006). Leishmania infantum LeIF protein
is an ATP-dependent RNA helicase and an eIF4A-like
factor that inhibits translation in yeast. FEBS J. 273,
5086–5100.
Nielsen, K. H., Chamieh, H., Andersen, C. B.,
Fredslund, F., Hamborg, K., Le Hir, H. & Andersen,
G. R. (2009). Mechanism of ATP turnover inhibition in
the EJC. RNA, 15, 67–75.
Dittrich, M. & Schulten, K. (2005). Zooming in on ATP
hydrolysis in F1. J. Bioenerg. Biomembr. 37, 441–444.
Petsko, G. A. (2000). Chemistry and biology. Proc. Natl
Acad. Sci. USA, 97, 538–540.
Gross, C. H. & Shuman, S. (1998). The nucleoside
triphosphatase and helicase activities of vaccinia virus
NPH-II are essential for virus replication. J. Virol. 72,
4729–4736.
Gavin, A. C., Aloy, P., Grandi, P., Krause, R., Boesche,
M., Marzioch, M. et al. (2006). Proteome survey reveals
modularity of the yeast cell machinery. Nature, 440,
631–636.
Tarassov, K., Messier, V., Landry, C. R., Radinovic, S.,
Serna Molina, M. M., Shames, I. et al. (2008). An in vivo
map of the yeast protein interactome. Science, 320,
1465–1470.
Yedavalli, V. S., Neuveut, C., Chi, Y. H., Kleiman, L. &
Jeang, K. T. (2004). Requirement of DDX3 DEAD box
RNA helicase for HIV-1 Rev-RRE export function.
Cell, 119, 381–392.
Zhang, M. & Green, M. R. (2001). Identification and
characterization of yUAP/Sub2p, a yeast homolog of
the essential human pre-mRNA splicing factor
hUAP56. Genes Dev. 15, 30–35.
Del Campo, M., Tijerina, P., Bhaskaran, H., Mohr, S.,
Yang, Q., Jankowsky, E. et al. (2007). Do DEAD-box
proteins promote group II intron splicing without
unwinding RNA? Mol. Cell, 28, 159–166.
965
51. Solem, A., Zingler, N. & Pyle, A. M. (2006). A DEAD
protein that activates intron self-splicing without
unwinding RNA. Mol. Cell, 24, 611–617.
52. Karow, A. R. & Klostermeier, D. (2009). A
conformational change in the helicase core is
necessary but not sufficient for RNA unwinding
by the DEAD box helicase YxiN. Nucleic Acids Res.
37, 4464–4471.
53. Chang, T. H., Latus, L. J., Liu, Z. & Abbott, J. M.
(1997). Genetic interactions of conserved regions in the
DEAD-box protein Prp28p. Nucleic Acids Res. 25,
5033–5040.
54. Bernstein, K. A., Granneman, S., Lee, A. V., Manickam, S. & Baserga, S. J. (2006). Comprehensive
mutational analysis of yeast DEXD/H box RNA
helicases involved in large ribosomal subunit biogenesis. Mol. Cell. Biol. 26, 1195–1208.
55. Granneman, S., Bernstein, K. A., Bleichert, F. &
Baserga, S. J. (2006). Comprehensive mutational
analysis of yeast DEXD/H box RNA helicases
required for small ribosomal subunit synthesis. Mol.
Cell. Biol. 26, 1183–1194.
56. Hotz, H. R. & Schwer, B. (1998). Mutational analysis of
the yeast DEAH-box splicing factor Prp16. Genetics,
149, 807–815.
57. Martin, A., Schneider, S. & Schwer, B. (2002). Prp43 is
an essential RNA-dependent ATPase required for
release of lariat-intron from the spliceosome. J. Biol.
Chem. 277, 17743–17750.
58. Plumpton, M., McGarvey, M. & Beggs, J. D. (1994). A
dominant negative mutation in the conserved RNA
helicase motif ‘SAT’ causes splicing factor PRP2 to
stall in spliceosomes. EMBO J. 13, 879–887.
59. Kim, D. W., Kim, J., Gwack, Y., Han, J. H. & Choe, J.
(1997). Mutational analysis of the hepatitis C virus
RNA helicase. J. Virol. 71, 9400–9409.
60. Tai, C. L., Pan, W. C., Liaw, S. H., Yang, U. C., Hwang,
L. H. & Chen, D. S. (2001). Structure-based mutational
analysis of the hepatitis C virus NS3 helicase. J. Virol.
75, 8289–8297.
61. Marintcheva, B. & Weller, S. K. (2001). Residues
within the conserved helicase motifs of UL9, the
origin-binding protein of herpes simplex virus-1, are
essential for helicase activity but not for dimerization
or origin binding activity. J. Biol. Chem. 276,
6605–6615.
62. Karginov, F. V., Caruthers, J. M., Hu, Y., McKay, D. B.
& Uhlenbeck, O. C. (2005). YxiN is a modular protein
combining a DEx(D/H) core and a specific RNAbinding domain. J. Biol. Chem. 280, 35499–35505.
63. Chen, Y., Potratz, J. P., Tijerina, P., Del Campo, M.,
Lambowitz, A. M. & Russell, R. (2008). DEAD-box
proteins can completely separate an RNA duplex
using a single ATP. Proc. Natl Acad. Sci. USA, 105,
20203–20208.
64. Liu, F., Putnam, A. & Jankowsky, E. (2008). ATP
hydrolysis is required for DEAD-box protein recycling
but not for duplex unwinding. Proc. Natl Acad. Sci.
USA, 105, 20209–20214.
65. Aregger, R. & Klostermeier, D. (2009). The DEAD box
helicase YxiN maintains a closed conformation during
ATP hydrolysis. Biochemistry, 48, 10679–10681.
66. Houk, K. N., Leach, A. G., Kim, S. P. & Zhang, X.
(2003). Binding affinities of host–guest, protein–
ligand, and protein–transition-state complexes.
Angew. Chem. Int. Ed. Engl. 42, 4872–4897.
67. Wentworth, P., Jr. & Janda, K. D. (2001). Catalytic
antibodies: structure and function. Cell Biochem.
Biophys. 35, 63–87.
966
68. Guthrie, C. & Fink, G. R. (1991). Guide to yeast
genetics and molecular biology. In Methods in Enzymology (Abelson, J. N. & Simon, M. I., eds), Methods in
Enzymology, vol. 194, pp. Academic Press, San Diego.
69. Mumberg, D., Muller, R. & Funk, M. (1995). Yeast vectors
for the controlled expression of heterologous proteins in
different genetic backgrounds. Gene, 156, 119–122.
Motif III in Superfamily 2 “Helicases”
70. Bommarito, S., Peyret, N. & SantaLucia, J., Jr.
(2000). Thermodynamic parameters for DNA
sequences with dangling ends. Nucleic Acids Res.
28, 1929–1934.
71. Turner, D. H., Sugimoto, N. & Freier, S. M. (1988).
RNA structure prediction. Annu. Rev. Biophys. Biophys.
Chem. 17, 167–192.