Bacillus subtilis DEAD protein YdbR possesses ATPase, RNA binding, and RNA unwinding activities

Article abstract of DOI:10.1271/bbb.50678

The product of an open reading frame (ORF) (called YdbR) identified while analyzing the Bacillus subtilis genome has been classified as an Asp-Glu-Ala-Asp (DEAD) protein, but the biological function and enzymology of YdbR have not been characterized in detail. Here we show that recombinant YdbR-His₆ purified from Escherichia coli is an ATP-independent RNA binding protein. It also possesses RNA-dependent ATPase activity stimulated not only by total RNA from B. subtilis but also by an RNA that is irrelevant to that of B. subtilis. Functional analysis indicated that the growth rate of a ΔydbR mutant strain of B. subtilis was reduced as compared with that of the wild type not only at 37°C, but more severely at 22°C.

Full text of DOI:10.1271/bbb.50678

Biosci. Biotechnol. Biochem., 70 (7), 1606–1615, 2006
Bacillus subtilis DEAD Protein YdbR Possesses ATPase,
RNA Binding, and RNA Unwinding Activities
y
Yoshinari ANDO and Kouji NAKAMURA
Graduate School of Life and Environmental Sciences, University of Tsukuba,
Tsukuba-shi, Ibaraki 305-8572, Japan
Received December 15, 2005; Accepted March 6, 2006; Online Publication, July 23, 2006
[doi:10.1271/bbb.50678]
The product of an open reading frame (ORF) (called
YdbR) identified while analyzing the Bacillus subtilis
genome has been classified as an Asp-Glu-Ala-Asp
(DEAD) protein, but the biological function and enzy-
mology of YdbR have not been characterized in detail.
Here we show that recombinant YdbR-His6 purified
from Escherichia coli is an ATP-independent RNA
binding protein. It also possesses RNA-dependent AT-
Pase activity stimulated not only by total RNA from
B. subtilis but also by an RNA that is irrelevant to that
of B. subtilis. Functional analysis indicated that the
growth rate of a ÁydbR mutant strain of B. subtilis was
reduced as compared with that of the wild type not only
at 37 ꢀC, but more severely at 22 ꢀC.
motifs (Ia, Ib, IV, and V) are probably involved in RNA
binding.7)
DEAD-box proteins are assumed to be ATP-depend-
ent RNA helicases, based on the in vitro RNA helicase
activity of the mouse translation initiation factor eIF-4A,
the human nuclear protein p68, Xenopus An3, and
Drosophila Vasa.8–12) Mutational analyses of eIF-4A
have further evidenced that its RNA unwinding activity
depends upon the hydrolysis of ATP. Moreover, the
Escherichia coli DEAD-box protein DbpA can destabi-
lize RNA duplexes in the presence of ATP,13) but some
DEAD-box proteins appear to be ATP-independent.
CsdA, a cold shock protein in E. coli, has been found to
unwind dsRNA in the absence of ATP.14)
Five genes encoding DEAD-box proteins have been
identified in E. coli: DbpA, CsdA, SrmB, RhlB, and
RhlE.13–17) Among these, DbpA, CsdA, and SrmB most
likely play a role in ribosome biogenesis.18–20) RhlB has
been found to be one component of the RNA degrado-
some, a multicomponent ribonucleolytic complex.21)
DbpA shares a highly conserved 70-amino acid C-
terminal extension common to several DEAD-box pro-
teins with YxiN,22) a protein encoded in the genome of
Bacillus subtilis.23) This homology indicates that YxiN
is a member of a sub-family that binds 23S rRNA and
functions in rRNA maturation/ribosome biogenesis or
in a currently unappreciated aspect of translation.
Indeed, a fragment of 23S rRNA specifically stimulates
the ATPase activity of YxiN, like that of DbpA.
Moreover, YxiN can unwind RNA duplexes.24)
In this study, we identified B. subtilis YdbR as the
second DEAD-box protein in B. subtilis. It contains a
central core region of nine conserved motifs that are
similarly spaced to those of DEAD-box proteins. Using
purified YdbR-His6, we found that YdbR has ATPase
activity in the presence of RNA and ATP-independent
RNA binding activity. Furthermore, YdbR unwound
RNA duplexes in the presence of ATP. In vivo YdbR
Key words: RNA helicase; Asp-Glu-Ala-Asp (DEAD)
protein; Bacillus subtilis; cold shock
There is increasing evidence that RNAs play crucial
roles in numerous processes in the cell. Structural
modulation of RNA is fundamental to the proper
execution of these processes, including translation and
ribosome biogenesis, as well as RNA splicing, matura-
tion, and degradation.1–3) Modulation of the RNA
secondary structure is catalyzed by a ubiquitous protein,
RNA helicase. Many putative RNA helicases have been
grouped into DEAD (Asp-Glu-Ala-Asp), DEAH (Asp-
Glu-Ala-His), or DExH (where x can be any amino acid)
box families based on their amino acid sequences.
DEAD-box proteins share a set of nine conserved amino
acid sequence motifs3) (Q-VI) (Fig. 1). The Q-motif and
motifs I and II (which are homologous to the Walker
A and B motifs4)) are responsible for the binding and
hydrolysis of ATP.5) Motif III is potentially involved in
coupling ATP hydrolysis to conformational changes
required for RNA-unwinding activity.6) Motif VI also
most likely participates in ATP binding.2) Structure
analyses of RNA helicases suggest that the remaining
y
To whom correspondence should be addressed. Tel: +81-29-853-6419; Fax: +81-29-853-7723; E-mail: nakamura.kouji@biol.tsukuba.ac.jp
Abbreviations: bp, base pair(s); DEAD, Asp-Glu-Ala-Asp; dsRNA, double-stranded RNA; DTT, dithiothreitol; eIF-4A, eukaryotic initiation factor-
4A; IPTG, isopropyl-1-thio-ꢀ-D-galactopyranoside; Ni-NTA, nickel-nitrilotriacetic acid; nt, nucleotide; ORF, open reading frame; PAGE, poly-
acrylamide gel electrophoresis; PCR, polymerase chain reaction; ssRNA, single-stranded RNA

Characterization of B. subtilis DEAD Protein YdbR
1607
Fig. 1. Alignment of Conserved Motifs in DEAD-Box RNA Helicases of B. subtilis and E. coli.
Sequences of YdbR (P96614) and YxiN (NP 391790) from B. subtilis, and RhlE (P25888), DbpA (P42305), SrmB (P21507), CsdA (P0A9P7),
and RhlB (P0A8J8) from E. coli were obtained from NCBI Genbank (accession nos. in parentheses).
protein was found to localize in the ribosome fraction
exclusively and to associate with 50S and 70S ribosomes
and polysomes. Depletion of YdbR resulted in a defect
in growth at 37 ꢀC and at low temperature (22 ꢀC),
suggesting that YdbR plays a critical role, interacting
with RNA substrates during translation at low temper-
ature.
NTA resin (Qiagen) and shaken gently for 60 min at
room temperature. Weakly bound proteins were re-
moved with buffer B (100 mM NaH2PO4, 10 mM Tris–
HCl, pH 6.3, 8 M urea). Proteins still bound to the resin
were finally eluted with buffer B, containing 0.2 M
imidazole. Fractions containing proteins were pooled
and sequentially dialyzed for 1 hour in buffer C (20 mM
Tris–HCl, pH 7.5, 0.5 M NaCl, 50% v/v glycerol, 2 mM
dithiothreitol, 1 mM EDTA, 0.1 mM phenylmethylsul-
phonyl fluoride (PMSF)), containing decreasing con-
centrations of urea-6 M, 4 M, 2 M, and 1 M, and then
dialyzed for over 12 h in buffer C, containing 0 M urea.
The concentration of the recombinant protein in the
solution was estimated to be about 4 mg/ml. This finding
was based on the standard curve obtained from the BCA
Protein Assay (Pierce Biotechnology, Rockford, IL)
using different concentrations of bovine serum albumin
(BSA). The estimated purity of the protein was over
95% based on laser densitometry of the Coomassie
Brilliant Blue stained gel using the NIH image program
(developed at the United States National Institutes of
Health). The data suggested that the recombinant YdbR-
His6 protein purified as described above from E. coli is
soluble.
Materials and Methods
Cloning, over-expression, and purification of YdbR.
To express YdbR with a hexa-histidine tag, we amplified
a DNA fragment encoding the entire region of the ydbR
gene by PCR using suitable primers: P1 (50-GATC-
CCATGGTAAATCACGACATTACTGAA-30) and P2
(50-GTACAGATCTGTAAGATTTTTTCTGGCGTCTG-
30); primer P1 carried a NcoI restriction site and primer
P2 a BglII site (shown underlined). The amplified DNA
was gel-purified, cleaved with NcoI and BglII, and
ligated into pQE60 (Qiagen, Tokyo, Japan) that also cut
with the two restriction enzymes. Six histidine codons
were added in frame into the plasmid for translation onto
the carboxyl terminus of the protein. Candidate clones
were transformed and verified by sequencing. E. coli
strain M15 harboring both pREP4 (Qiagen) and the
constructed plasmid was cultured on LB plates in the
presence of 50 mg/ml ampicillin. pREP4 confers resist-
ance to kanamycin, and it constitutively expresses the
lac repressor protein encoded by the lacI gene. A single
colony was inoculated into 5 ml of LB liquid medium
containing 100 mg/ml ampicillin and 10 mg/ml kanamy-
cin and incubated for 16 h at 37 ꢀC. The cultured cells
were then inoculated into 100 ml of fresh LB liquid
medium and vigorously shaken for 2 h. After IPTG
(2 mM final concentration) was added, the cells were
incubated for an additional 5 h. The His-tagged protein
was then purified as described previously.25) The cells
were pelleted by centrifugation and resuspended in
buffer A (100 mM NaH2PO4, 10 mM Tris–HCl, pH 8.0,
8 M urea). The suspension was disrupted by gentle
mixing for 3 min, followed by stirring for 10 min at
room temperature. The lysates were separated by
centrifugation at 10;000 ꢁ g for 20 min at 4 ꢀC to pellet
the cellular debris. The supernatant was added to Ni-
Preparation of RNA substrates. We prepared an RNA
substrate from a DNA fragment containing the T7 RNA
polymerase promoter and multi-cloning sites of pGEM-
3zf (+) (Promega, Madison, WI). A 207 bp fragment
corresponding to the region between 60 bp upstream and
146 bp downstream of the transcription initiation site
on pGEM-3zf (+) was obtained by PCR amplification
with the primers (50-GTTTTCCCAGTCACGACGTT-30
and 50-GAATTGTGAGCGGATAACAA-30). We then
prepared a 147-nucleotide RNA by in vitro transcription
with T7 RNA polymerase (Takara Bio, Ohtsu, Japan)
using the amplification fragment as described above.
The RNA was labeled for filter-binding assay by adding
[ꢁ-32P] CTP (400 Ci/mmol, Amersham Biosciences,
Little Chalfont, United Kingdom) to the in vitro tran-
scription reaction. RNA-I (50-GAAUACUCAAGCU-
UG-30) was complementary to the region between
positions 55 and 69 of the 147-nucleotide RNA tran-
scribed in vitro. To prepare 50- and 30-overhanging

1608
Y. ANDO and K. NAKAMURA
substrates, RNA-II (50-UAUAGUGAGUCUUAU-30)
and RNA-III (50-AGCUCGAAUUCGCCC-30), were
chemically synthesized. RNA-II and -III are comple-
mentary to the regions from positions 133 to 147 and 1
to 15 respectively of the 147 nucleotide RNA. These
chemically synthesized RNAs were end-labeled with T4
polynucleotide kinase (Toyobo, Osaka, Japan) using [ꢂ-
32P] ATP (6,000 Ci/mmol, Amersham Biosciences). To
allow the formation of an RNA–RNA hybrid, 147-
nucleotide RNAs synthesized in vitro and 32P-labeled
RNAs were heated in buffer H (20 mM Tris–HCl, pH
7.5, 100 mM KCl) at 95 ꢀC for 5 min. After cooling to
4 ꢀC over 90 min, samples were separated into controls
for hybridization and to monitor unwinding activity.
RNA-IV (50-GGGCGAAUUCGAGCU-30) was comple-
mentary to RNA-III to generate blunt-end RNA–RNA
duplex. In this case, RNA-III was 32P-labled and
hybridized to RNA-IV.
GTP was assayed, ATP was replaced by 1 mCi [ꢂ-32P]
GTP (6,000 Ci/mmol, Amersham Biosciences) in the
reaction mixture. Observed values were converted to
mmol [ꢂ-32P] released.
RNA helicase activity assays. The standard RNA
helicase assay was conducted in a total volume of 20 ml
containing 20 mM Tris–HCl, pH 7.5, 100 mM NaCl,
3 mM MgCl2, 2 mM DTT, 100 mg/ml BSA, 3 pmol
RNA–RNA partial duplex, 150 pmol of the YdbR-His6
protein, and 1 mM ATP unless otherwise indicated.
Reactions were initiated by adding the indicated amount
of YdbR-His6 protein. After incubation at 35 ꢀC for
60 min, reactions were stopped with 5 ml of loading
buffer (0.1 M Tris–HCl, pH 7.5, 20 mM EDTA, pH 7.5,
0.5% w/v SDS, 0.1% v/v NP-40, 50% v/v glycerol,
0.1% w/v BPB). Control samples lacked YdbR-His6
protein (total duplex), or contained a partial RNA–RNA
duplex that was heated at 95 ꢀC for 5 min, and then
rapidly chilled on ice (total single-stranded RNA).
Samples were separated on SDS–10% w/v polyacryl-
amide gels in 9 mM Tris-borate, pH 8.3, 2 mM EDTA,
and 0.1% w/v SDS. Gels were dried and exposed to X-
ray film (Fuji Photo Film, Tokyo, Japan) for 12 h. The
intensity of each band was normalized to the total
intensity of each lane and is expressed as the percentage
of total duplex or total single-stranded RNA.
Nitrocellulose filtration assay. We incubated 32P-
labeled RNA probes (20 nM) for 1 minute at room
temperature with various concentrations of purified
YdbR-His6 (0.001–1 mM) in 10 ml of buffer D (20 mM
Tris–HCl, pH 7.4, 10 mM MgCl2, 50 mM NaCl, 0.3%
v/v vanadium ribonucleoside complex, 1 unit of poly
(dI-dC)). Nitrocellulose filters (0.45-nm pore size; Toyo
Roshi, Tokyo, Japan) were washed with washing buffer
(20 mM Tris–HCl, pH 7.4, 10 mM MgCl2, 100 mM NaCl,
10% v/v glycerol). The mixture, containing purified
YdbR-His6, was then diluted with 0.5 ml of washing
buffer and immediately passed through filters with
1.5 ml of the same buffer. The levels of radioactivity
remaining on the filters were determined using a liquid
scintillation counter (Beckman Coulter, Fullerton, CA).
Sucrose density gradient centrifugation. Vegetative
cells growing at OD600 of 0.5 were collected by
centrifugation and resuspended in a buffer E (20 mM
Tris–HCl, pH 7.6, 15 mM Mg(CH3COO)2, 100 mM
CH3COONH4, 6 mM ꢀ-mercaptoethanol, 2 mM PMSF),
and disrupted by passage through a French pressure cell
(Otake Works, Tokyo, Japan) at 20,000 p.s.i.27) After
removal of cell debris, the supernatant was subjected
to 10–40% sucrose density gradient centrifugation
(SW41Ti rotor, Beckman Coulter: 40 A260 unit per
tube) in buffer E at 22,000 rpm for 15 h. After centri-
fugation, 500 ml aliquots were collected from the top of
the gradient (a total of 20 fractions) using a Piston
Gradient Fractionator (BioComp Instruments, Frederic-
ton, Canada). We measured the absorbance of each
fraction at 260 nm, and then added 125 ml of 50%
trichloroacetic acid (TCA) to each fraction, mixed them
well, and placed the solution on ice for 30 min. The
aggregated proteins were precipitated by centrifugation,
washed in cold acetone, dried, and dissolved in the
sample solution (0.4 M Tris–HCl, pH 6.8, 2% w/v SDS,
0.5% v/v ꢀ-mercaptoethanol, 10% v/v glycerol).
ATPase activity assays. ATPase activity was deter-
mined using 10 nM YdbR-His6 by hydrolysis of ATP to
ADP and phosphate in a reaction mixture of 1ꢁ ATPase
buffer (20 mM Tris–HCl, pH 7.5, 100 mM NaCl, 3 mM
MgCl2, 2 mM DTT, 1 mCi [ꢂ-32P] ATP). ATPase stock
solutions were adjusted to pH 7.5 prior to use. Assays
were carried out with and without transcript, either
synthesized in vitro using pGEM-3zf (+) linearlized at
the HindIII site with SP6 RNA polymerase (Takara Bio)
or total RNA fraction prepared from B. subtilis, as
described previously.26) The reaction mixture (10 ml)
was incubated at 37 ꢀC for 10, 20, and 30 min and
stopped by the addition of 200 ml of 70% w/v activated
charcoal powder (Merk, Whitehouse Station, NJ) sus-
pended in 50 mM HCl and 5 mM H3PO4. Samples were
mixed and then centrifuged at 15;000 ꢁ g for 10 min at
room temperature. Levels of radioactivity released into
the supernatant were measured using a liquid scintilla-
tion counter. The background radioactivity was deter-
mined in a similar reaction without protein and
subtracted from all other reactions. All assay points
were collected in duplicate and corrected against a
buffer blank. When the ability of YdbR to hydrolyze
Western blot analysis of YdbR protein. Proteins were
separated on SDS–PAGE and then electroblotted onto
polyvinylidene difluoride (PVDF) membranes (Milli-
pore, Billerica, MA). Non-specific binding was blocked
with 5% w/v skim milk in buffer F (20 mM Tris–HCl,
pH 7.5, 154 mM NaCl, 0.2% v/v Tween 80), and
proteins were then probed using rabbit anti-B. subtilis

Characterization of B. subtilis DEAD Protein YdbR
1609
YdbR serum (1:10,000 dilution in buffer F) for 60 min.
After probing, the membrane was rinsed with buffer F
and then incubated with secondary antibody (donkey
anti rabbit IgG) for 60 min. The membranes were
washed with buffer G (20 mM Tris–HCl, pH 7.5,
154 mM NaCl, 0.5% v/v Tween 80), and bound antibody
was detected using enhanced chemiluminescence
(Amersham Biosciences).
Construction of the ydbR deletion strain and in vivo
depletion of YdbR. The 50 region of the B. subtilis ydbR
gene encoding the amino acids from residues 7 to 107 of
YdbR was amplified by PCR (restriction sites are
underlined: 50-GATCAAGCTTGACGAGCAGCAAG-
CAT-30 and 50-GTACAGATCTAGTACTCAGTACA-
TATA-30) from B. subtilis genomic DNA. The 322-bp
PCR product was trimmed with HindIII and BglII and
ligated into pMutin228) previously digested with HindIII
and BamHI. E. coli strain C600 was transformed with
the recombinant plasmid on LB-ampicillin agar plates
to convey ampicillin resistance. The sequence of the
YdbR-pMutin2 construct was verified by DNA sequenc-
ing. This new plasmid, named pTUE2002, was used
to transform B. subtilis strain 168trpC2 to create
erythromycin (0.3 mg/ml) resistance, providing B. sub-
tilis ꢃydbR. Correct integration of a single-copy of ydbR
gene into the plasmid was confirmed by Southern blot
analysis.
Results
Identification and heterologous expression of YdbR
We isolated a novel DEAD-box protein, YdbR, in
B. subtilis, based on its conserved amino acid se-
quences. The in vivo function of this 511-amino acid
protein (58.1 kDa predicted molecular mass) is un-
known. YdbR contained the DEAD motif in addition
to eight other conserved amino acid motifs (Fig. 1),
suggesting that it is a DEAD-box protein. The gene
encoding YdbR was amplified from B. subtilis chromo-
somal DNA by PCR, sequenced, and cloned into
the pQE60 expression vector to generate plasmid
pTUE2002-1. M15 (pREP4) cells were transformed
with pTUE2002-1, and the YdbR-His6 protein was
expressed at high levels with an estimated homogeneity
of over 95%.
Fig. 2. RNA Binding Activity and ATPase Activity of Recombinant
YdbR Protein.
A, RNA binding activity of recombinant YdbR. RNA binding
activity of YdbR was measured by filter assays in the presence (open
circle) or absence (closed circle) of ATP, as described under
‘‘Materials and Methods.’’ Retention of YdbR-RNA complexes on
nitrocellulose filters was monitored. RNA bound was defined as
follows: percent RNA retained = retained labeled on filter (cpm) ꢁ
100/added labeled RNA (cpm). Values were plotted against added
YdbR concentrations (mM). B, Time course of recombinant YdbR
ATP hydrolysis activities. ATPase activity is expressed as mM
(concentrations) of released inorganic phosphate in 10 ml reaction
volumes. ATPase activity was measured in the presence (open
circles) or absence (closed circles) of B. subtilis total RNA, and the
presence of the run-off transcribed RNA using linearized pGEM-3zf
(+) plasmid as template (open square). C, Steady state kinetics of
YdbR ATPase. The ATPase activities (mM/min/mM protein) of the
purified YdbR were assayed at various concentrations of B. subtilis
total RNA. ATPase activities were expressed as mM (concentrations)
of 32Pi released from [ꢂ-32P] ATP during a 1-min incubation at
37 ꢀC, and are plotted as functions of input RNA. Each data point
represents the average of three independent determinations.
Characterization of YdbR RNA-binding and ATPase
activities
We assayed the ability of YdbR to bind RNA using
nitrocellulose filtration. YdbR-His6 protein bound RNA
even when derived from a pGEM-3zf (+) plasmid
(Fig. 2A). Moreover, no radioactivity was observed on
the filter when protein was passed over it, followed by
the radiolabeled RNA, indicating that membrane-bound
protein cannot trap RNA (data not shown). YdbR
binding activity did not depend on the presence of
ATP. The binding affinity (M1=2) of YdbR-His6 to RNA,

1610
Y. ANDO and K. NAKAMURA
defined as the RNA concentration required to give half-
maximal binding, was 0.1 mM.
We tested the biochemical function of the purified
YdbR-His6 by measuring its ability to hydrolyze ATP,
and examined whether this activity depended on the
presence of RNA, a characteristic feature of the DEAD-
box family. We measured the ATPase activity of the
purified YdbR-His6 protein to release radioactive phos-
phate from [ꢂ-32P] ATP. As Fig. 2B shows, YdbR-His6
hydrolyzed ATP in the presence of total B. subtilis RNA
as well as in that of transcripts derived from pGEM-3zf
(+) plasmid. No difference between the rates of ATPase
activity in presence of B. subtilis total RNA and pGEM-
derived RNA was observed (4 ATP minꢂ1 YdbRꢂ1). No
activity was observed in the absence of RNA (Fig. 2B).
Moreover, the ATPase activity was eliminated when
ribonucleaseA was added to the reaction (data not
shown), indicating that it was dependent on the added
RNA. Thus YdbR-His6 exhibited RNA-dependent AT-
Pase activity, and neither single- nor double-stranded
DNA stimulated its activity (data not shown). Experi-
ments were performed using varying RNA concentra-
tions at saturating ATP levels (Fig. 2C), as well as by
varying the ATP concentration at saturating RNA (data
not shown). The average Km (ATP) value for YdbR-
His6, determined in three independent experiments, was
calculated to be 225 mM. On the other hand, the Km
(ATP) value for eukaryotic eIF-4A is 190 mM and E. coli
DbpA is 120 mM, and it is known that the Km (ATP) of
DEAD-box proteins ranges from 50 to 500 mM.3,18,29)
Thus it appears that the Km (ATP) of YdbR-His6 is
consistent with that of other DEAD-box proteins. As
shown in Fig. 2C, the ATPase activity of YdbR-His6
increased as RNA was added. As deduced from the data
in Fig. 2A, almost all of the YdbR-His6 in a reaction
binds to RNA, suggesting that the majority of YdbR-
His6 protein in our protein stock was active. We also
examined whether YdbR-His6 can hydrolyze other
nucleotide triphosphates. Under the conditions used to
demonstrate ATP hydrolysis by YdbR-His6, radioactive
phosphate was not detectably released in the presence of
ꢂ-32P-labelled GTP (data not shown), indicating that this
protein cannot hydrolyze GTP.
Fig. 3. RNA Helicase Activity of Recombinant YdbR Protein.
RNA unwinding assays proceeded as described in ‘‘Materials and
Methods.’’ A, Structure of artificial duplex RNA substrate. Synthetic
32P-labeled RNA is indicated by an asterisk (ꢃ). B, Influence of
YdbR concentration on RNA unwinding activity. RNA helicase
reactions proceeded in the presence of various amounts of YdbR in
20 ml reactions at 35 ꢀC for 60 min. Lane 1, substrate RNA alone;
lane 2, substrate RNA heated and immediately chilled on ice. Lanes
3–10, 5, 10, 30, 55, 75, 100, 125, and 150 pmol of YdbR
respectively. Positions of RNA substrate and single RNA are
indicated (‘‘a’’ and ‘‘b’’). C, ATP dependent helicase activity of
YdbR. RNA helicase reactions preceded using 150 pmol of YdbR in
the presence of 1 mM nucleotide triphosphate (lane 1, ATP; lane 2,
no triphosphate; lane 3, GTP; lane 4, CTP; lane 5, UTP; lane 6,
ATP ꢂ-S). In lane 7, the unwinding assay was performed with
150 pmol of YdbR with ATP (1 mM) and 25 mM EDTA. Lane ‘‘—’’
contained no protein.
Characterization of RNA unwinding activity
Because sequence homology with other DEAD-box
proteins suggested that YdbR is an RNA helicase, we
investigated its ability to destabilize RNA–RNA hybrids
using a standard dsRNA substrate consisting of 147- and
15-base RNAs. 15-base RNA hybridized to a central
15-base sequence of 147-base RNA to form a dsRNA
(Fig. 3A). This substrate contained both 50 and 30 single-
stranded regions, and only the 15-base RNA was labeled
with 32P. The dsRNA substrate migrated to position ‘‘a’’
(Fig. 3B, lane 1). When the substrate was incubated at
95 ꢀC followed by quick cooling, the un-annealed 15-
base RNA labeled with 32P migrated to position ‘‘b’’
(Fig. 3B, lane 2). Increasing the concentration of YdbR-
His6 in the assay mixture containing dsRNA and ATP
(1 mM) caused band ‘‘b’’ to appear (Fig. 3B, lanes 3–10).
This indicates that the amount of dsRNA substrate
unwinding increased. A dose-response curve indicated
that 16.2 pmol/20 ml of YdbR-His6 protein was required
to unwind 50% of the input substrate at 37 ꢀC (data not
shown). A helicase assay was performed at saturating
concentrations of YdbR-His6 (54.68 pmol/20 ml), indi-

Characterization of B. subtilis DEAD Protein YdbR
1611
Fig. 4. Subcellular Localization of YdbR.
A, Sedimentation profile showing free subunits, 70S ribosomes, and polysomes from the B. subtilis strain 168trpC2 cells. Cells were grown at
37 ꢀC to OD600 of 0.2 in liquid LB medium and harvested. Cell extracts were separated from the top of 10–40% sucrose gradients into 20
fractions of 500 ml, and each fraction was continuously monitored at A260. Sedimentation is from left to right. B, Detection of YdbR and L2. The
total proteins were prepared from equal volumes of each fraction (nos. 1 to 18), resolved on SDS–12% polyacrylamide gels, and Western blotted.
YdbR and L2 in each lane were detected using anti-YdbR and anti-L2 antiserum respectively.
cating that the observed helicase activity is a time-
dependent process (data not shown).
the RNA helicase activity of YdbR-His6 is dependent on
ATP. The presence of EDTA diminished RNA unwind-
To examine the unwinding polarity of YdbR-His6
protein, 50-overhang, 30-overhang, and blunt-end RNA
duplexes were tested (data not shown). Unwinding was
observed among duplexes with 50 and 30 overhangs,
indicating that YdbR-His6 can catalyze bidirectional
unwinding, but blunt-end RNA duplexes did not serve as
a substrate. Quantification analysis revealed that only
10% of blunt-end RNA duplex was unwound, indicating
that a single-strand overhang stimulates YdbR-His6
unwinding activity.
To determine the specificity of the energy source
in the RNA unwinding reactions, we tested all four
nucleotide triphosphates (ATP, CTP, GTP, and UTP)
and the non-hydrolysable ATP analog, ATP-ꢂ-S
(Fig. 3C). Consistently with the notion that YdbR-His6
is an ATP-dependent RNA helicase, this protein
obviously preferred ATP. In the absence of ATP, no
unwinding activity was evident (Fig. 3C, lane 2). No
activity was observed when the other three NTPs (GTP,
UTP, and CTP), or ATP ꢂ-S were included in the
reaction, although YdbR-His6 still bound RNA in their
presence (Fig. 3C, lane 3–6). These results indicate that
ing activity, suggesting that the reaction is Mg2þ
dependent (Fig. 3C, lane 7).
-
Subcellular localization of YdbR on a sucrose density
gradient centrifugation
Many DEAD-box proteins are involved in translation
initiation or ribosome biogenesis.1,3,30) Examination of
protein localization gives a useful suggestion for
elucidation of its function. To examine whether YdbR
is co-localized with ribosomes in B. subtilis cells, cell
extracts prepared from B. subtilis wild-type strain
168trpC2 at the exponential phase were separated by
sucrose density gradient centrifugation (Fig. 4A). Pro-
teins from each fraction were precipitated by TCA and
separated on SDS–PAGE, and YdbR in each fraction
was detected by immunoblotting using rabbit anti-YdbR
IgG (Fig. 4B). Assignments of the 70S ribosome and
50S subunit were made to detect B. subtilis L2 protein
(Fig. 4B) by immunoblotting using anti-serum raised
against L2. Quantitative analysis of the density of each
band, shown in Fig. 4B, indicated that over 95% of the
total YdbR sedimented with 50S subunits, 70S ribo-

1612
Y. ANDO and K. NAKAMURA
Fig. 5. Effects of YdbR Depletion on Growth of B. subtilis Strains 168trpC2 (wt) and ꢃydbR at Different Temperatures.
Growth curves of wt (open circles) and ꢃydbR (closed circles) at 37 ꢀC (A), 45 ꢀC (B), and 22 ꢀC (C). Cells were incubated in liquid LB
medium, and growth was monitored at OD600
.
somes, and polysomes (Fig. 4B, lanes 13, 17 to 20, and
22), suggesting that YdbR associates with ribosomes.
The association of YdbR with ribosomes was slightly
sensitive to high salt concentrations. When 300 mM
potassium chloride was added to the cell extracts, 80–
90% of the YdbR that was associated with ribosomes
became dissociated (data not shown). This result
suggests that this protein associates with ribosomal
proteins through ionic interactions rather than rRNAs.
strain in liquid medium was slow and inhibited as
compared to wild-type growth (Fig. 5A), but at a high
temperature (45 ꢀC) the influence of YdbR depletion
disappeared (Fig. 5B). Exponential growth of the mutant
cells at 37 ꢀC was significantly impaired as compared to
wild-type cells, which grew twice as fast as the mutant.
The growth rate of the ꢃydbR strain progressively
slowed to a doubling time of 84 min during the
exponential growth phase, compared with 30 min for
the wild-type strain (Fig. 5A). Under these conditions,
no YdbR protein was detected in cell extracts prepared
from the ꢃydbR strain (data not shown). Moreover, the
ꢃydbR strain showed a cold-sensitive phenotype on
growth at a lower temperature (22 ꢀC, Fig. 5C). The
doubling time during the exponential growth phase of
the ꢃydbR strain was 183 min at 22 ꢀC, compared to
93 min for the wild-type strain. These results indicate
Effects of YdbR depletion on cell growth
To evaluate the function of YdbR in vivo, a 50
terminal portion of a genomic copy of the ydbR gene
was deleted by homologous recombination to yield
strain ꢃydbR. The growth rates of the wild-type
(168trpC2) and ꢃydbR strains were compared at differ-
ent temperatures. At 37 ꢀC, the growth of the ꢃydbR

Characterization of B. subtilis DEAD Protein YdbR
1613
that YdbR is more important for a normal growth rate
during the exponential phase at 22 ꢀC than at 37 ꢀC.
required only below 30 ꢀC. The deletion of ydbR caused
a decrease in 50S subunits on ribosome profiles (Ando
and Nakamura, unpublished), but differed from srmB
and csdA deletion mutants, which result in the accumu-
lation of 23S rRNA precursor and a new particle
sedimenting around 40S.19,20) Moreover, deletion of
ydbR did not affect the composition of ribosomal
proteins (Ando and Nakamura, unpublished). As Fig. 5
shows, YdbR is required for vegetative growth at 37 ꢀC
and is more important for growth at 22 ꢀC. The
secondary structures of RNA are more stable at lower
temperatures and might pose a problem during recog-
nition of mRNA translation initiation sites by ribosomes
or elongation reaction of mRNA translation. Based on
these data, we propose that YdbR affects translation
efficiency by modifying secondary structures of RNA at
low temperatures.
Three cold-shock-induced RNA helicases have been
reported: CsdA in E. coli,14) CrhC in the cyanobacte-
rium Anabaena sp. strain PCC 7120,33) and DeaD in
Antarctic archaeon Methanococcoides burtonii.34) These
proteins are specifically induced on a downshift in
temperature but are not expressed at significant levels at
their respective optimal growth temperatures. Moreover,
the csdA deletion mutant specifically affected growth at
low temperatures, but no such effect was observed at its
optimal growth temperature.10) On the other hand, YdbR
showed high expression during the vegetative phase at
37 ꢀC, and the expression of this protein was enhanced
by a temperature downshift (Ando and Nakamura,
unpublished). Moreover, growth of the ꢃydbR strain
was inhibited not only at 37 ꢀC (Fig. 5A), but, more
severely, at 22 ꢀC (Fig. 5C).
Discussion
We identified a novel B. subtilis DEAD-box RNA
helicase, YdbR. The amino acid sequence, including
central core regions with nine conserved motifs, is
aligned with E. coli RNA helicases in Fig. 1. This
protein sequence homology along with the spacing of
the nine conserved motifs is significant, and we
predicted that YdbR is a DEAD-box RNA helicase.
The protein was expressed in E. coli, purified, and
biochemically characterized in terms of its enzymatic
properties. With respect to ATPase activity, that of
purified YdbR was RNA-dependent, and no activity
was detected in the absence of RNA. E. coli DbpA is
activated only by a specific region of 23S rRNA that
contains part of the peptidyltransferase center of the
ribosome.18) The B. subtilis DEAD-box protein, YxiN,
is also activated by an analogous region of 23S rRNA
(no. 2481–2634).22) As shown in Fig. 3B, both the RNA
derived from the pGEM-3zf (+) plasmid and the total
RNA from B. subtilis stimulated the ATPase activity of
YdbR. The RNA derived from the in vitro transcription
using the pGEM-3zf (+) plasmid as a template did not
contain a characteristic secondary structure. Moreover, it
did not have significant sequence homology with the
rRNAs in B. subtilis. These results suggest that YdbR
prefers the sequence included in the in vitro tran-
scription and in the total RNA fraction. To determine
the character of substrate RNAs of YdbR, an experi-
ment, such as SELEX (Systematic Evolution of Ligands
by EXponential enrichment) must be done.
The helicase was fully active in a standard assay using
RNA substrate, which contains a 15 bp duplex flanked
by ssRNA at both the 50 and the 30 end. Moreover, YdbR
demonstrated ATP-dependent RNA unwinding activity
in the absence of accessory proteins. As Fig. 3C shows,
this unwinding activity requires ATP hydrolysis. YdbR
can unwind a dsRNA duplex in both the 50- and the 30-
direction. Moreover, the helicase activity of YdbR
requires ssRNA tails for its activity. Although YdbR
and eIF-4A are ATP-dependent helicases, the features of
YdbR RNA helicase activity differ from those of human
eIF-4A, which is able to unwind blunt-ended duplexes,
as well as 50- and 30-overhang RNA duplexes.31) On the
other hand, the hepatitis C virus non-structural protein 3
(NS3) catalyses unidirectional 30 to 50 unwinding of
RNA and RNA/DNA duplexes.32)
We propose that YdbR is the first DEAD-box RNA
helicase required for efficient growth, especially at a low
temperature (22 ꢀC). At low temperatures, the secondary
structure of RNA is stabilized, and it is expected that the
function of YdbR, which induces structural changes of
mRNA 50 UTR of genes, is necessary for efficient
translation.
Acknowledgments
We thank Dr. Kozo Ochi of the National Food
Research Institute of Japan for kindly providing the
antiserum raised against B. subtilis ribosomal protein.
And we thank Dr. Yorgo Modis of Yale University for
critical reading of the manuscript. This work was
supported by Grants-in-Aid for Scientific Research (B)
from the Japan Society for the Promotion of Science
(JSPS) and by Grants-in-Aid for JSPS Fellows.
We observed that YdbR interacts with free 50S
subunits, 70S ribosomes, and polysomes, as shown in
Fig. 4. CsdA and SrmB, which are found as DEAD-box
RNA helicases in E. coli, are involved in the ribosome
biogenesis of 50S ribosomal subunits, as previously
reported by Charollais et al.19,20) SrmB affects 50S
assembly at an early stage and CsdA is involved in 50S
assembly at a later stage than SrmB, but CsdA is
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