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INTRODUCTION |
DEAD-box proteins form a large family of putative RNA helicases
that show sequence similarity to eIF4A, a eukaryotic translation initiation factor. All members of the DEAD-box family share eight conserved amino acid motifs, including the characteristic sequence Asp-Glu-Ala-Asp (DEAD in the single-letter code) that inspired their
name (1). Sequence comparisons identified the closely related DEAH- and
DExH-box families, which together with the DEAD-box proteins, form the
helicase superfamily II (2). The DEAH and DExH families notably include
DNA helicases involved in DNA replication and recombination. The
putative RNA helicases of superfamily II are found in a wide range of
organisms, including bacteria, viruses, and eukaryotes ranging from
yeast to humans. Although they are involved in very diverse cellular
functions, such as pre-mRNA splicing, rRNA processing, and mRNA
export, translation, and decay, they are all supposed to share in
common an RNA helicase activity (3, 4). This activity has been inferred
from the ability of eIF4A to melt out mRNA structure (5) or to
dissociate an RNA duplex in vitro (6), in an
ATP-dependent manner. However, although NTPase activity has
been demonstrated for all purified DEAD-box and related proteins, RNA
helicase activity has been characterized for only a few of them and
remains conjectural in most cases.
Nevertheless, numerous steps of gene expression are likely to require
RNA helicase activity, either to unwind RNA secondary structures or to
rearrange large RNA structures, or even to disrupt RNA-protein
interactions. For example, transient base pairings between small
nuclear RNAs and between small nuclear RNAs and pre-mRNA, which
occur during pre-mRNA splicing, are often mutually exclusive and
thus need to form and dissociate sequentially. At least eight DEAD-box
and related proteins have thus far been shown to be required for
splicing in yeast and may accomplish these structural rearrangements
(7). Similarly, 13 DEAD-box are assumed to be involved in extensive
rearrangements between pre-rRNA and ribosomal proteins/small nucleolar
RNAs during ribosome biogenesis (8).
Translation initiation in eukaryotes also presumably requires removal
of secondary structure in the 5'-untranslated region of mRNAs for
the binding of the small ribosomal subunit and its migration toward the
AUG codon ("scanning") (9). Indeed, insertion of stable stem-loop
structures in the 5'-untranslated region of mRNAs inhibits
translation initiation in both higher eukaryotes (e.g. 10, 11) and yeast (e.g. 12, 13). In mammals, unwinding of these
structures has been attributed to the cap-binding complex eIF4F,
because it shows an RNA helicase activity in vitro (6), and
its overexpression in vivo facilitates translation of
mRNAs with highly structured 5'-untranslated regions (14). This
complex consists of eIF4E, which binds to the cap structure at the
5'-end of mRNAs, eIF4G, and eIF4A, which is the active helicase
component. Another factor, eIF4B, may also be involved in the unwinding
process because it is required for the helicase activity of eIF4A
in vitro (6, 15).
Despite the high degree of conservation of translation in eukaryotes,
the cap-binding complex in the yeast Saccharomyces
cerevisiae is not equivalent to mammalian eIF4F, because yeast
eIF4A is not found in the complex (16). Moreover, yeast eIF4A has been
shown to be active in RNA unwinding in vitro with mammalian
eIF4B (17), but not with its putative yeast counterpart, Tif3p (18),
suggesting that additional yeast factors are required to catalyze this
reaction. We and others (19, 20) have recently described another
DEAD-box protein, Ded1p, required for translation initiation in
S. cerevisiae. We isolated the DED1 gene as a
multicopy suppressor of a temperature-sensitive mutation in eIF4E.
Analyses of its suppressor activity, of polysome profiles of
ded1 mutant strains, and of synthetic lethal interactions with different translation initiation mutants indicated that Ded1p has
a role in translation initiation (19). Consistently, immunodepletion of
Ded1p in an in vitro translation system abolished
translation activity (20). Genetic data suggest that Ded1p and eIF4A
play independent roles in translation initiation, but the nature of these roles is yet unknown. Here, we investigate the biochemical activities of Ded1p and show that it has RNA-dependent
ATPase and ATP-dependent RNA helicase activities, both
activities depending on the integrity of the DEAD motif. RNA binding
studies also show that the affinity of Ded1p for RNA can be modulated
by ATP and ADP, suggesting how RNA unwinding may be driven by ATP hydrolysis.
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EXPERIMENTAL PROCEDURES |
Strains--
The S. cerevisiae strains used in this
study are derivatives of W303 (MATa/MAT
ura3-1/ura3-1
ade2-1/ade2-1 his3-11,
15/his3-11, 15 leu2-3,
112/leu2-3, 112 trp1-1/trp1-1). The strain ID2
(MATa/MAT
DED1/ded1::HIS3MX6) was obtained by
disrupting one DED1 open reading frame copy with the HIS3MX6
marker module (21) in W303. ID2-2A (MAT
ded1::HIS3MX6) is a meiotic segregant of ID2 that
requires a plasmid-borne copy of DED1 for viability. The
inability of ID2-2A to lose the resident YCplac33-DED1
plasmid (19) was tested by plasmid shuffling (22), using
5-fluoro-orotic acid plates. Standard yeast genetic techniques and
media were as described (23).
Expression and Purification of Ded1p--
To express an
N-terminal His6-tagged Ded1 fusion protein from its cognate
promoter, a fusion polymerase chain reaction was performed (24).
Briefly, two fragments with sequence overlap were generated in a first
polymerase chain reaction series with YCplac33-DED1 as a
template and the oligonucleotide couples 5'-GCAGAGGCTAGCAGAATTAC-3' (5'-DED1 homology region, external
oligonucleotide)/5'-GTGATGGTGGTGATGGTGCATATGAATATGAAATGCTTTTCTTGTTG-3' (the DED1 homology region is in boldface, and the
overlapping part containing the 6-histidine tag is underlined; an
NdeI site has also been introduced (italics)), and
5'-CATATGCACCATCACCACCATCACGCTGAACTGAGCGAACAAG-3' (same conventions as above; the DED1 sequence starts at the
second codon)/5'-GGCAACGATAGGGACGGAG-3' (3'-DED1 homology
region, external oligonucleotide) as primers. The polymerase chain
reaction products, together with the external primers, were used for
the fusion polymerase chain reaction. The final product was cloned as a
PstI-EagI fragment into the
PstI-EagI-restricted YCplac111-DED1
(19) to yield YCplac111-His6-DED1. This
construct complemented the ded1 null allele to the wild-type extent. The NdeI-EcoRI fragment of
YCplac111-His6-DED1 plasmid was cloned into
NdeI-EcoRI-restricted pET-22b. The resulting
pET22b-His6-DED1 plasmid was transformed into
MO20-1, a BL21(DE3) derivative of Escherichia coli that is
deficient in RNaseE activity (25). Transformed cells were grown and
induced with 0.5 mM
isopropyl-1-thio-
-D-galactopyranoside for 3 h.
Then, they were harvested, resuspended in 20 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 5 mM imidazole, and lysed
by sonication. After centrifugation (30 min at 40,000 × g), the supernatant was loaded on a 1-ml
nickel-nitrilotriacetic acid-agarose column (Qiagen). After two washes
with 30 and 60 mM imidazole, the His6-Ded1p was eluted with 250 mM imidazole. The eluted fractions were
adjusted to 50% glycerol and stored at
20 °C. We usually obtained
around 0.6 mg of Ded1p from 200 ml of culture. The protein has a
tendency to precipitate; where necessary, precipitated protein was
removed by centrifugation.
Construction of the DAAD Mutant--
To change the glutamic acid
residue of the DEAD motif into alanine, the
pET22b-His6-DED1 plasmid was mutagenized
in vitro with the QuikChange site-directed mutagenesis kit
of Stratagene. A 613-nt-long BamHI-BstBI fragment
from candidates carrying the desired mutation was entirely sequenced
and cloned into the BamHI-BstBI-restricted original pET22b-His6-DED1 plasmid to eliminate
eventual mutations outside the fragment. The mutant protein was
purified as for the wild-type. The NdeI-SalI
fragment from the mutated pET22b-His6-DED1 plasmid was cloned into the NdeI-SalI-restricted
YCp111-His6-DED1 plasmid.
ATPase Assay--
ATPase activity was monitored continuously by
a coupled spectrophotometric method (26). This method uses pyruvate
kinase and lactate dehydrogenase to link hydrolysis of ATP to oxidation of NADH, which results in a decrease in the absorbance at 338 nm.
Assays were performed at 37 °C in a reaction volume of 0.4 ml, in
buffer containing 20 mM Tris-HCl, pH 8.0, 50 mM
KCl, 5 mM MgCl2, 1 mM
dithiothreitol, 1 mM ATP, 300 µM NADH, 2 mM phosphoenolpyruvate, and 3 units/ml of pyruvate kinase
and lactate dehydrogenase. RNA and protein were added as indicated in
Figs. 2 and 3. Absorbance data were analyzed using Kaleidagraph 3.0 (Synergy). The steady-state rate of ATP hydrolysis equals that of NADH
oxidation, which was quantified using 6300 M
1
cm
1 for the extinction coefficient of NADH.
RNA Preparations--
Total RNA from W303 yeast strain was
extracted by the acid-phenol method (27). Poly(A)+
(polyadenylated) RNA was prepared from total RNA using an oligo(dT) column. The 18 and 25 S rRNAs were prepared from 40 and 60 S ribosomal subunits that were separated by centrifugation on a sucrose gradient as
described in Ref. 19. Their purity was checked by gel electropohoresis. Poly(A), poly(C), poly(G), and poly(U) homopolymers and yeast tRNA were
purchased from Sigma.
RNA substrates used for helicase and RNA binding assays were prepared
as described (28). Briefly, two transcripts of 43 and 68 nucleotides
were synthesized in vitro from pGEM-3Z and pGEM MO1/2
vectors, the shorter one being labeled with [
-32P]GTP
(specific activity, 2.5 × 106 cpm/pmol). The nucleotide
sequences of the two strands are as follows: 43-nt strand,
5'-GAAUACUCAAGCUUGCAUGCCUGCAGGUCGACUCUAGAGGAUC-3'; 68-nt
strand,
5'-GGGAGACCGGAAUUCCCCAUGGCUGACUAAUUUUUUUUAUUUAUGCAGAGGGGGGAUCCUCUAGAGUC-3' (duplex region is underlined). These transcripts were annealed and
the resulting partial duplex, which consists of a 14-base pair region
flanked by 5' ss1 overhangs
of 29 and 54 nucleotides (see Fig. 4A), was purified by
native polyacrylamide gel electrophoresis. Its specific activity was
calculated to be 2.5 × 106 cpm/pmol. We also
constructed a 55-base pair "complete" duplex by annealing a 64-nt
RNA synthesized by SP6 RNA polymerase from EcoRI-restricted
pGEM3 plasmid (labeled with [
-32P]GTP) and a 60-nt RNA
synthesized by T7 RNA polymerase from Hind III-restricted
pGEM3 plasmid.
RNA Helicase Assay--
Reaction mixtures (15 µl) contained 20 mM Tris-HCl, pH 8.0, 70 mM KCl, 2 mM MgCl2, 2 mM dithiothreitol, 15 units RNasin, 45 fmol (3 nM) of partial duplex, and, where
indicated, 2 mM ATP. Reactions were initiated by addition
of Ded1p, as indicated in Fig. 4. After incubation for 10 min at
37 °C, the reactions were stopped by the addition of 4 µl of a
solution containing 1.2% SDS, 10 mM EDTA, 40% glycerol,
bromphenol blue, xylene cyanol, and 250 µg/ml proteinase K. For
positive controls (cf. Fig. 4, lane 1), duplex
RNA was separated into monomers by heating at 95 °C for 2 min,
followed by rapid cooling in ice. Samples were electrophoresed on a
10% polyacrylamide gel in 0.5× TBE. Labeled RNAs were visualized by
autoradiography and quantified using a Fuji Bas 1000 phosphorimager.
The percentage of unwinding was calculated using the formula
((monomer/total)
(monomerø/totalø)) × 100, where
"total" is the amount of monomer plus duplex, and ø refers to the
numbers observed in absence of ATP.
RNA Filter Binding Assay--
RNA substrates for the binding
reactions were either the duplex used in the helicase assay, or the
labeled 43-nt transcript used to construct this duplex, or, in some
experiments, the complete duplex. Reaction mixtures (20 µl) contained
20 mM Tris-HCl, pH 8.0, 70 mM KCl, 2 mM MgCl2, 2 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, 40 fmol (2 nM) of RNA, and
varying concentrations of Ded1p. When present, nucleotides (ADP and
AMP-PNP (Sigma)) were used at a final concentration of 2 mM. Samples were incubated for 5 min at room temperature
before filtering. A "double-filter" assay was performed following
the method of Ref. 29, using a dot-blot apparatus. The RNA-protein
complexes were retained on nitrocellulose (Schleicher & Schuell),
whereas free RNA was retained on a charged nylon membrane
(Hybond-N+, Amersham Pharmacia Biotech) placed underneath.
Membranes were washed before and after loading the samples with 1 ml of
ice-cold reaction buffer. The amount of RNA present on each membrane
was quantified using a Fuji Bas 1000 phosphorimager, and the fraction of RNA bound to the protein was determined. All assays were corrected for the small fraction of bound RNA in absence of protein (on average,
1.5%). Binding curves were obtained by plotting the fraction of bound
RNA versus protein concentration.
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RESULTS |
Purification of Ded1p--
To facilitate the biochemical
characterization of Ded1p, we overexpressed it in E. coli as
a fusion protein with a 6-histidine tag inserted at its N terminus. To
test the functionality of the modified Ded1 protein (hereafter called
His6-Ded1p) in S. cerevisiae, the fusion gene
under its own promoter was cloned into a yeast centromeric plasmid
(YCp, low copy number). The resulting plasmid (YCplac111-His6-DED1) or a control plasmid
harboring the untagged DED1 gene (YCplac111-DED1)
was transformed into strain ID2-2A (YCplac33-DED1). Upon
plasmid shuffling on 5-fluoro-orotic acid plates and subsequent
restreaking on YPD plates, His6-DED1
complemented the ded1 null allele to the wild-type extent at
all temperatures tested (16, 30, and 37 °C), indicating that the
modified Ded1p was functional in vivo (data not shown).
The His6-DED1 gene was then placed under the T7
promoter, and the resulting plasmid
(pET22b-His6-DED1) was introduced into a
derivative of the E. coli strain BL21(DE3). The
His6-Ded1p was overexpressed after
isopropyl-1-thio-
-D-galactopyranoside induction (Fig.
1). Although a small portion was
insoluble, most of the His6-Ded1p was found in the soluble
fraction of cell extracts. The recombinant protein was purified on a
nickel-agarose column and was about 90% pure as judged on a Coomassie
Brilliant Blue-stained acrylamide gel (Fig. 1). The apparent molecular
mass of Ded1p (about 68 kDa) coincides with that estimated by sequence
analysis. The identity of the protein was verified by Western blot
analysis using anti-polyhistidine antibodies (data not shown). The
polypeptides that co-eluted are most likely E. coli
contaminating proteins rather than degradation products, because they
also appeared in eluates of E. coli extracts that harbored
pET22b without the DED1 gene.

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Fig. 1.
Expression and purification of
His6-Ded1p. Histidine-tagged Ded1p was overproduced in
E. coli and purified by affinity on a nickel resin column.
M, protein size markers; , total proteins from uninduced
cells; +, total proteins from
isopropyl-1-thio- -D-galactopyranoside-induced cells;
Ni, fraction containing His6-Ded1p after elution
(250 mM imidazole) from nickel column. Proteins were
resolved by SDS-polyacrylamide gel electrophoresis and stained with
Coomassie Brilliant Blue.
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To analyze the importance of the DEAD motif on Ded1p properties, we
constructed a mutated allele in which the highly conserved glutamic
acid residue was substituted for an alanine. The DEAD and DExH motifs
are variants of the Walker ATPase B motif and the corresponding
aspartic and glutamic acid residues have been shown to participate in
ATP hydrolysis (15). The mutated DED1 allele was inserted
into YCplac111, which was then transformed into the ID2-2A
(DED1-URA3) strain. The transformants were unable to grow on
5-fluoro-orotic acid medium, showing that the mutated protein is not
functional in vivo. This protein, hereafter called the DAAD
mutant, was overexpressed in E. coli and purified as for the
wild-type protein. The yield and purity were similar to those obtained
with the wild-type; in particular, the same contaminating polypeptides
were observed.
Ded1p Has an RNA-dependent ATPase Activity--
The
ATPase activity was measured continuously by a spectrophotometric
method in which ATP hydrolysis is coupled to NADH oxidation, which is
itself monitored by measurement of A338 nm (see
under "Experimental Procedures"). Fig.
2 represents typical raw data generated
by the ATPase assay. In the absence of RNA, no change of
A338 nm could be detected, indicating that Ded1p
has no ATPase activity by itself. In the presence of total yeast RNA,
the absorbance decreased linearly with time due to the oxidation of
NADH until all NADH was exhausted. The slope of the decrease is
proportional to the rate of ATP hydrolysis. Increasing Ded1p
concentration 4-fold resulted in a 4-fold increase of this rate.
Addition of RNase A during the reaction immediately abolished the
activity, confirming that it was dependent on the added RNA (data not
shown). Thus, Ded1p exhibits an RNA-dependent ATPase
activity characteristic of members of the DEAD-box family. From kinetic
analyses performed in the presence of saturating concentration of total
yeast RNA, we determined the Km for ATP to be around
300 µM and the Vmax to be 5-10
µmol of ATP/min/mg of protein, which corresponds to a turnover number
of 340-680 µmol of ATP/min/µmol of protein.

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Fig. 2.
ATPase activity of Ded1p. ATPase
activity of wild-type or mutant Ded1p (DAAD) was measured by a
spectrophotometric assay in the presence or absence of total yeast RNA.
Oxidation of NADH, which is directly proportional to the rate of ATP
hydrolysis, was continuously monitored by measuring the absorbance at
338 nm. , Ded1p (147 nM) without RNA; , Ded1p (38 nM) with RNA (50 µg/ml); , Ded1p (147 nM)
with RNA (50 µg/ml); , DAAD Ded1p (74 nM) with RNA (50 µg/ml).
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The DAAD mutant showed a 60-fold reduced rate of ATP hydrolysis,
indicating that the DEAD motif is important for the ATPase activity. It
also excluded the possibility that the activity observed with the
wild-type protein was due to minor E. coli contaminants.
To test for a possible specificity of the ATPase reaction toward the
RNA substrate, we compared a variety of RNAs in the assay, using
saturating RNA and ATP concentrations in each case. As shown on Fig.
3, most RNAs tested activated the ATPase
activity, but at different levels. The highest activity was observed
with poly(A)+ mRNAs and rRNAs, whereas homopolymers and
tRNA were far less efficient. Total RNA from E. coli also
stimulated the ATPase activity; in contrast, DNA, whether ss or ds, was
inactive in this assay.

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Fig. 3.
Stimulation of Ded1p ATPase activity by
various substrates. The y axis represents the rate of
ATP hydrolysis (in µmol of ATP hydrolyzed/min/µmol of protein).
Each reaction contained 30 nM Ded1p and either 20 µg/ml
RNA (total or poly(A)+ mRNA from yeast, 25 S and 18 S
yeast rRNA), 50 µg/ml homopolymer RNA (poly(A), poly(U), poly(C),
poly(G)), or 200 µg/ml yeast tRNA; the concentrations of each RNA
species were previously determined to induce maximal ATPase activity.
DNA (50 µg/ml) was either from S. cerevisiae or E. coli.
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Ded1p Has ATP-dependent RNA Helicase Activity--
To
test whether Ded1p has RNA helicase activity, we examined its ability
to displace a partial duplex RNA (called "duplex"), as in Ref. 6.
To this end, we constructed a standard substrate consisting of a 68-nt
RNA annealed over 14 nucleotides to a shorter radiolabeled 43-nt RNA
(Fig. 4A). As shown in Fig.
4B, the duplex migrated much slower than the 43-nt ssRNA
(compare lanes 2 and 1). The reactions contained
45 fmol (3 nM) of the RNA duplex and were incubated for 10 min at 37 °C with different amounts of Ded1p. The helicase activity
was monitored by determining the amount of 43-nt ssRNA (called
"monomer") released from the duplex. In a reaction mixture
containing a large excess of Ded1p, but no ATP, no duplex dissociation
was detected (lane 2). In the presence of ATP, the amount of
monomer increased with increasing concentrations of Ded1p (lanes
3-8), showing that Ded1p has an ATP-dependent helicase activity. Quantification of the duplex and monomer showed that
90% of the duplex was dissociated in presence of 50 nM
protein, but dissociation was weaker with lower concentrations (see the legend of Fig. 4). Thus, a 15-fold excess of protein was required for
nearly complete dissociation. The reason for this requirement is
discussed below.

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Fig. 4.
RNA unwinding activity of Ded1p.
A, scheme of the partial duplex. B, the RNA
unwinding activity was tested by the ability of the protein to
dissociate the partial duplex. Forty-five fmol (3 nM) of
labeled substrate were incubated with increasing concentrations of
Ded1p or with the DAAD mutant in the presence or absence of 2 mM ATP. The products of the reactions were separated by a
10% native polyacrylamide gel electrophoresis and visualized by
autoradiography. Lane 1, duplex was boiled before loading on
the gel; lane 2, Ded1p without ATP; lanes 3-8,
increasing amounts of Ded1p in the presence of ATP resulted in 2%
(lane 3), 12% (lane 4), 56% (lane
5), 68% (lane 6), and 90% (lanes 7 and
8) unwinding; lanes 9-10, DAAD mutant without
and with ATP does not unwind the duplex.
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We also measured the unwinding activity as a function of time and found
that in the presence of excess protein (80 nM), maximal dissociation was reached within 1-2 min (data not shown).
The DAAD mutant, which is deficient for ATPase activity, was unable to
dissociate the duplex (Fig. 4B, lanes 9 and 10);
moreover, the wild-type protein did not dissociate the duplex when ATP
was replaced by AMP-PNP, a nonhydrolyzable analog of ATP (data not shown). These results indicate that helicase activity requires ATP hydrolysis.
Characterization of the RNA Binding Activity of Ded1p--
To
investigate further the unwinding activity of Ded1p and the role played
by ATP hydrolysis, we examined whether ATP and ADP influence the
binding of Ded1p to RNA. We used a filter binding assay in which
RNA-protein complexes are retained by a nitrocellulose membrane and
free RNA by a charged nylon membrane (see under "Experimental Procedures"). The substrates used were either a duplex of 55 base pairs containing ss 5'-ends of only 5 and 9 nucleotides (called complete duplex), the partial duplex substrate used in the helicase assay, or its 43-nt monomer. The ability of Ded1p to bind these species
was determined by incubating a fixed amount of RNA (40 fmol, 2 nM) with increasing amounts of Ded1p and measuring the amount of protein-bound RNA in each case. Fig.
5A shows that whereas Ded1p
had a marked affinity for the monomer (the majority of the RNA
molecules were retained on the filter for Ded1p concentrations above
100 nM, with an average Kd of 20 nM), it bound to the complete duplex extremely weakly, if
at all (no more than 1% of the input RNA was retained even at the
highest Ded1p concentration used). In contrast, Ded1p could bind to the
partial duplex, suggesting that this binding can be partially or
totally ascribed to the long 5' ss extensions of this molecule.
However, the percentage of input RNA bound was much lower than with the
monomer, particularly at low Ded1p concentrations. Because, even at the
highest concentrations used, no more than 35% could be bound, it is
difficult to decide whether the majority of the partial duplex
molecules bind Ded1p with an extremely weak affinity, or whether only a
subpopulation is responsible for the weak binding.

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Fig. 5.
RNA binding activity of Ded1p. The
ability of Ded1p (A) and its DAAD mutant (B) to
bind either to the monomer ( ) or to the partial duplex ( ) was
measured by nitrocellulose filter binding. Binding of the wild-type
protein to the complete duplex (see text) was also assayed ( ). Forty
fmol (2 nM) of labeled RNA were incubated with increasing
concentrations of protein. The percentage of RNA bound is plotted
against the concentration of protein. The dashed curves
represent the best fit to the equation RNAb = RNAmax [Ded1p]/(Kd + [Ded1p]),
where RNAb is the fraction of RNA bound at each
protein concentration and RNAmax is the maximal fraction of
RNA bound. The data are from two or three independent
measurements.
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We also measured the RNA binding capacity of the DAAD mutant protein.
As shown in Fig. 5B, the mutant bound to RNA with nearly the
same affinity as the wild-type. Thus, ATP hydrolysis is not required
for Ded1p binding to RNA. Similar results have been reported for other
RNA helicases (30-32).
We then analyzed the effects of nucleotides (ATP and ADP) on the RNA
binding properties of the wild-type Ded1 protein (Fig. 6). To avoid ATP hydrolysis and duplex
unwinding during the measurements, we used a nonhydrolyzable analog of
ATP, AMP-PNP. To check that the binding of AMP-PNP mimics that of ATP,
we tested its ability to compete with ATP in the ATPase assay, using
subsaturating ATP concentrations (half the determined
Km). Equimolar and 5- and 10-fold excesses of
AMP-PNP with respect to ATP, resulted in 50, 75, and 80% inhibition of
the ATPase activity, respectively (not shown). Therefore, AMP-PNP can
compete with ATP in the ATPase reaction and thus presumably binds to
Ded1p similarly. Now, whereas AMP-PNP had a modest effect on the
binding of Ded1p to the monomer (the maximal percentage of bound RNA
remained nearly the same, and the apparent Kd
decreased slightly to 12 nM), it affected much more
drastically the binding to the partial duplex. Even at low Ded1p
concentrations, the percentage of RNA bound was now quite appreciable,
exceeding half the value observed with the monomer, and at high Ded1p
concentration, it reached a plateau that was consistently above that
observed with the monomer (Fig. 6A). We conclude that
AMP-PNP greatly increases the affinity of Ded1p for the partial duplex
molecules and possibly also the proportion of these molecules that can
be bound. In contrast, ADP had almost no effect on the binding of the
protein to either partial duplex or monomer (Fig. 6B). In
summary, the affinity of Ded1p for the partial duplex varies markedly
depending on which nucleotide is present. These results suggest that
hydrolysis of ATP to ADP modulates the relative affinities of Ded1p for
ssRNA and dsRNA. As discussed below, this modulation might be
responsible for the unwinding activity.

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Fig. 6.
Effect of nucleotides on Ded1p RNA binding
activity. The binding of Ded1p to monomer ( ) and partial duplex
( ) RNAs was measured in presence of either AMP-PNP (A) or
ADP (B). Experimental conditions were as in Fig. 5.
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DISCUSSION |
In this work, we have shown that the putative RNA helicase Ded1p,
a DEAD-box protein essential for translation initiation in S. cerevisiae, is a bona fide RNA helicase. It possesses
an RNA-dependent ATPase activity, an RNA unwinding activity
that depends on ATP hydrolysis, and a differential binding to ssRNA and
dsRNA that is modulated by ADP and the ATP analog AMP-PNP.
ATPase Activity--
The ATPase activity of Ded1p is highly
stimulated by natural RNAs, such as rRNAs and polyadenylated mRNAs,
whereas homopolymers are far less efficient. Among the DEAD-box
proteins that have been studied biochemically, only DbpA from E. coli shows a strong RNA substrate specificity (33, 34). Other
characterized DEAD-box and related proteins either do not show any
substrate specificity in the ATPase assay or show a specificity that
seems unrelated to their assumed in vivo role. For example,
mammalian eIF4A, which is required for translation initiation of all
mRNAs, is more effectively stimulated by poly(U) than by globin
mRNA (35). Similarily, the Upf1 protein (superfamily I helicase
family), which is involved in nonsense-mediated mRNA decay in
yeast, is stimulated by homopolymers but not by total RNA (36). Thus,
the physiological substrates of these proteins cannot always be
inferred from their in vitro preferences. Nevertheless, the
fact that polyadenylated mRNAs and rRNAs are the best stimulators
of the ATPase activity of Ded1p suggests that Ded1p may interact with
these RNAs in vivo, consistent with its general role in
mRNA translation (19, 20).
We have obtained a Km value for ATP of around 300 µM, i.e., similar to that reported for other
DEAD-box proteins, such as p68, yeast eIF4A, and DbpA. This value does
not denote particularly tight ATP binding, but it stands below the
cellular ATP level (1-10 mM), indicating that Ded1p can
bind and hydrolyze ATP in the cell cytoplasm. The specific activity of
Ded1p (turnover number of 340-680 min
1) is higher than
that obtained for eIF4A (3 min
1 for the mammalian factor
(37) and 6.8 min
1 for the yeast factor (17)), SrmB (1.2 min
1) (38), p68 (45 min
1) (39), RNA
helicase II (1.9 min
1) (40), RNA helicase A (54 min
1) (41), Prp16p (90 min
1) (42), and An3
(6 min
1) (43), and it is similar to that of Upf1p (490 min
1) (36), DbpA (600 min
1) (34) and Prp22p
(400 min
1) (44). Thus, Ded1p has a higher ATPase activity
than many DEAD-box and related proteins for which this activity has
been measured.
Although the DAAD mutant is as efficient as the wild-type for binding
RNA, its ATPase activity is 60-fold lower, implying that the mutation
impairs ATP binding and/or hydrolysis. Interestingly in this respect,
mutational analyses performed with other helicases have shown that the
conserved aspartic and glutamic acid residues are necessary for ATP
hydrolysis but not binding (15, 45, 46). We suggest that, analogously,
the DEAD motif of Ded1p is directly implicated in ATP hydrolysis.
RNA Helicase Activity--
We have shown that purified Ded1p
exhibits RNA unwinding activity in vitro. Among the 39 DEAD-box and related proteins of S. cerevisiae, only 4 have
been shown to possess RNA unwinding activity: eIF4A (17); Dbp5p, a
DEAD-box involved in poly(A)+ RNA export (47); and Prp16p
(48) and Prp22p (44, 49), two DEAH-box proteins involved in splicing.
Upf1p, a superfamily I protein, has also been shown to possess RNA
unwinding activity (36). Whereas Prp16p, Prp22p, and Upf1p are active
without other proteins, eIF4A needs the assistance of mouse eIF4B, and
Dbp5p seems to require a still unknown cofactor. In contrast, our
observation that Ded1p purified from E. coli can unwind an
artificial template on its own indicates that it has an intrinsic
unwinding activity.
A Molar Excess of Protein over RNA Is Required for Efficient RNA
Unwinding--
We have found that high concentrations of Ded1p (
50
nM, i.e. a large excess with respect to the
template) are needed for efficient unwinding. This result raises the
possibility that unwinding is not a true enzymatic reaction; for
example, Ded1p could passively unwind the duplex simply by occupying
the ssRNA appearing due to thermal fluctuations. Such a stoichiometric
"dissociating" activity has been attributed to DbpA and CsdA, two
DEAD-box proteins of E. coli, which do not require ATP for
duplex dissociation (50, 51). However, in the case of Ded1p, ATP is
required for the unwinding activity, and AMP-PNP cannot substitute for
it; moreover, the DAAD mutant, which binds to ss and duplex RNAs as
does the wild-type protein but is deficient for the ATPase activity, is also deficient for unwinding. Thus, ATP hydrolysis is necessary for
driving the unwinding reaction, arguing that this reaction is a
catalytic rather than a stoichiometric process. In addition, we note
that the unwinding reaction only takes place in the presence of ATP,
i.e. under conditions where the binding preference of Ded1p
for ss compared with duplex RNA is precisely minimal (Fig. 6A).
Why, then, is Ded1p required in such a large excess for efficient
unwinding? Although we do not exclude that our Ded1p preparation may
not be 100% active, we believe that this requirement reflects the
reversibility of the reaction. Indeed, we have observed spontaneous reannealing (25-40%) when denatured duplex was incubated under standard conditions in the absence of Ded1p (results not shown). An
excess of Ded1p would then be necessary for maximizing the fraction of
the template that binds the protein, thereby increasing the unwinding
rate and, ultimately, displacing the reaction toward dissociation. We
speculate that, in vivo, smaller amounts of Ded1p may be
required if RNA-binding proteins bind to the ssRNA during unwinding,
preventing reassociation. Such a role has been attributed to the
E. coli ssDNA-binding protein, which has been shown to stimulate the activity of some DNA helicases (e.g. 52, 53).
The requirement for an excess protein over RNA is not unique to Ded1p;
rather, it has been observed for most RNA helicases studied so far
(e.g. Prp16p (48), Prp22p (44), RNA helicase II (40), Upf1p
(36), eIF4A (6, 15), p68 (39)). Among the RNA helicases characterized,
only NPH-II (54) and the human RNA helicase A (41) could act in
catalytic amounts, being able to dissociate a 10-fold molar excess of
RNA duplexes. These helicases may have particularly high turnovers for
the unwinding reaction; alternatively, with the particular substrates
used, the dissociated strands might fold intramolecularly, preventing
their reassociation.
What Could be the Mechanism of Unwinding?--
Like most DEAD-box
and related proteins, Ded1p requires an ss region for binding to RNA,
as shown by its inability to bind to a duplex containing very short
tails. Therefore, its affinity for the partial duplex probably reflects
mainly binding to the ss flanking regions. In the presence of AMP-PNP,
this affinity increases to nearly the same level as for ssRNA, possibly
reflecting the fact that Ded1p now binds to the ds part of the duplex
or to the ss-ds junction. In the presence of ADP, the affinity of Ded1p
becomes again higher for ssRNA. These results suggest that ATP binding
and hydrolysis modulate the relative affinities of Ded1p for ssRNA
versus dsRNA. Although further mechanistic studies are
required to understand the coupling between ATPase and helicase activities, this affinity modulation presumably plays a key role in the
unwinding process. We hypothesize that a conformational change of Ded1p
occurs upon ATP hydrolysis, leading to a preferential binding to ss and
hence to the separation of the two strands. Consistently, DNA
helicases, which have been characterized extensively with respect to
their DNA binding properties, have been shown to couple binding and
hydrolysis of nucleotides to conformational changes that alter their
affinity for different forms of DNA, thus driving the reaction (55). In
particular, Wong and Lohman (56) have shown that binding of ADP to Rep
dimer favors a state that binds preferentially to ssDNA, whereas in the
presence of a nonhydrolyzable analog of ATP, both ss and duplex DNA
were bound, a situation similar to that observed here. These authors
proposed an active "rolling" mechanism, in which the active form of
Rep is a dimer with both subunits binding alternatively to duplex and
ssDNA at an ssDNA-dsDNA junction. Alternative models for unwinding, which do not necessitate helicase oligomerization, have also been proposed (see Ref. 57).
In contrast to DNA helicases, mechanistic studies with RNA helicases
are still at their initial stages. However, Lorsch and Herschlag (37,
58) have shown recently that ATP binding and hydrolysis produce a cycle
of conformational changes in mammalian eIF4A that modulates its
affinity for ssRNA. Further work is needed to establish whether all DNA
and RNA helicases use the same mechanism for coupling ATP hydrolysis
and unwinding.
Role of the Ded1p RNA Helicase in Translation
Initiation--
Translation initiation in eukaryotes involves many
RNA-RNA, protein-RNA, and protein-protein interactions that have to be disrupted as translation elongation starts, presumably with the help of
RNA helicases. Until now, the only protein involved in translation
initiation that has been shown to possess RNA helicase activity, is
eIF4A. Although current views propose that eIF4A unwinds secondary
structures in 5'-mRNA untranslated regions, its precise function is
still elusive. Moreover, yeast eIF4A requires a still unknown cofactor
for RNA helicase activity. We show here that Ded1p, another DEAD-box
protein that is required for translation initiation in yeast, functions
as an RNA helicase in vitro on its own. Its ATPase and RNA
helicase activities are necessary for its function in vivo
because a mutation that abolishes them leads to lethality. Because
DED1 has been isolated as a multicopy suppressor of a mutant
of the cap-binding protein eIF4E, Ded1p might be involved in the
unwinding of cap-proximal secondary structures, perhaps to facilitate
cap recognition. More generally, genetic interactions suggest that
Ded1p plays a role at the level of ribosome binding (19). It remains to
be determined how its RNA helicase activity participates in this
biological function.