From the Institute of Biological Sciences, University of Tsukuba, Tsukuba-shi, Ibaraki 305, Japan
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ABSTRACT |
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Small cytoplasmic RNA (scRNA) is metabolically
stable and abundant in Bacillus subtilis cells. Consisting
of 271 nucleotides, it is structurally homologous to mammalian signal
recognition particle RNA. In contrast to 4.5 S RNA of Escherichia
coli, B. subtilis scRNA contains an Alu
domain in addition to the evolutionarily conserved S domain. In this
study, we show that a 10-kDa protein in B. subtilis cell
extracts has scRNA binding activity at the Alu domain. The
in vitro binding selectivity of the 10-kDa protein shows
that it recognizes the higher structure of the Alu domain of scRNA caused by five consecutive complementary sequences in the two
loops. Purification and subsequent analyses demonstrated that the
10-kDa protein is HBsu, which was originally identified as a member of
the histone-like protein family. By constructing a HBsu-deficient
B. subtilis mutant, we showed that HBsu is essential for
normal growth. Immunoprecipitating cell lysates using anti-HBsu antibody yielded scRNA. Moreover, the co-precipitation of HBsu with
(His)6-tagged Ffh depended on the presence of scRNA,
suggesting that HBsu, Ffh, and scRNA make a ternary complex and that
scRNA serves as a functional unit for binding. These results
demonstrated that HBsu is the third component of a signal recognition
particle-like particle in B. subtilis that can bind the
Alu domain of scRNA.
The first step of secretory pathway needs protein factors that
distinguish secretory proteins from cytoplasmic proteins. In higher
eukaryotes, the first distinction between secreted and cytoplasmic
proteins occurs at the ribosome upon the specific association of
SRP1 with the signal sequence
(1, 2). SRP purified from canine pancreas is composed of a 7 S RNA (7 SL RNA, referred to here as SRP RNA) and six proteins (SRP9, SRP14,
SRP19, SRP54, SRP68, and SRP72) (3). cDNAs for each protein have
been cloned (4-9). The SRP proteins are associated with the RNA as
either monomers (SRP19 and SRP54) or heterodimers (SRP9/SRP14 and
SRP68/SRP72). SRP54 is the best characterized component of SRP. SRP54
interacts with both SRP RNA and signal sequences and binds GTP
(10-12). Studies using SRP constituted with either a subset of the SRP
proteins or components modified with N-methyl maleimide indicate that
SRP19 is required for SRP54 to associate with SRP RNA, SRP9/SRP14 is required for elongation arrest, and modification of SRP68/SRP72 by
N-methyl maleimide prevents the close interaction of SRP with SRP
receptor and inhibits translocation-promoting activity (13). On the
other hand, the targeting of bacterial preproteins to the inner
membrane also seems to involve other cytoplasmic pathways that converge
at the membrane SecYEG translocon (14-18). One pathway unique to
Escherichia coli cells involves a secretion-specific chaperone like SecB. This chaperone guides preproteins, such as proOmpA, to a membrane-associated receptor, SecA, which provides a link
to the translocon complex (19). A second targeting pathway in
prokaryotes involves chaperones that are not specific for secretory proteins. Roles for these chaperones become important when normal targeting pathways are impaired. DnaJ and DnaK are required for the
residual transport of SecB-dependent proteins (20).
In addition to these pathways, a third pathway has been proposed
because components of the SRP and SRP receptor have been identified in
a wide variety of species (21). In Bacillus subtilis, scRNA,
Ffh, and Srb (FtsY), which are homologous to eukaryotic SRP RNA, SRP54,
and the SRP RNAs have been identified in all cells analyzed to date. Larsen and
Zwieb (29) proposed that mammalian SRP RNA consists of eight helices
(numbered 1-8). Mammalian SRP RNA (7SL RNA) is subdivided into two
domains. The Alu domain, consisting of helices 1-5 (helix 1 is unique to B. subtilis and Archaebacteria), seems to have
arose during evolution to the Alu interspersed repetitive sequences found in the human genome (30); the S domain, comprising part
of helix 5 and helices 6-8, contains 150 nucleotides of core sequence
lacking Alu repeat similarity. An evolutionary comparison has revealed that almost all Archaebacteria and eukaryotes, except yeast, contain SRP RNA consisting of eight helices. The nucleotide sequence in the region corresponding to helix 8 is highly conserved among SRP RNA homologues. On the other hand, the Alu domain
contains the two smallest subunits, SRP9 and SRP14, and their sequences comprise about 100 nucleotides from the 5' end and 50 nucleotides from
the 3' end of SRP RNA, respectively. Moreover, phylogenetic analyses
indicate that SRP RNA is the evolutionary progenitor of the repetitive
Alu sequences (31). Functional analysis of mammalian SRP RNA
demonstrated that the Alu domain is required for elongation
arrest, because SRP particles lacking this domain are deficient in this
function (13). In contrast to the structural integrity observed among
eukaryotes and Archaebacteria SRP RNAs, the length and secondary
structure of eubacterial SRP RNA vary. E. coli 4.5 S RNA
consists of 114 nucleotides that can fold into a single hairpin
corresponding to helix 8 and part of helix 5 (32). Almost all SRP RNA
from Gram-negative bacteria have a secondary structure similar to that
of E. coli (29). On the other hand, the secondary structure
of SRP RNAs from Gram-positive bacteria differs from that of E. coli. Bacillus subtilis scRNA is considered to be a member of the
SRP RNA family (22). It is transcribed as a 354-nucleotide precursor
and then processed to a 271-nucleotide RNA at both the 5' and 3'
cleavage sites by ribonuclease III (Bs-RNaseIII) (33, 34). B. subtilis scRNA is predicted to fold into a structure that is
strikingly similar to that of eukaryotic SRP RNAs, although it lacks
helices 6 and 7. Functional analyses have shown that scRNA depletion
leads to defects in the production of extracellular enzymes and in
Here we identified B. subtilis HBsu (10 kDa) protein with
respect to its binding affinity for the Alu domain of scRNA
(referred to as the scRNA Alu domain) that consists of
helices 1-4 and part of helix 5. Using several mutants of scRNA, we
found that the HBsu protein recognizes the secondary structure of these
helices rather than the specific nucleotide sequences. We also show
that HBsu, Ffh, and scRNA make a stable complex in vivo and
that scRNA functions as a backbone for complex formation. We discuss
the structural similarity of HBsu to that of the SRP9/SRP14 heterodimer demonstrated by Birse et al. (36).
Bacterial Strains and Growth Conditions--
Table
I lists the plasmids and bacterial
strains used. The E. coli and B. subtilis strains
were cultivated in Luria Bertani medium. Antibiotics were added at the
following concentrations: chloramphenicol, 5 µg/ml; ampicillin, 50 µg/ml; and tetracyclin 10 µg/ml. IPTG was added at 2 mM
unless otherwise indicated.
Plasmid Construction--
Mutant sc103 is thought to have
helices 1-5. Nucleotides 80 and 249 of sc103 are linked with the
sequence GGAA, a structurally well-defined GNRA tetraloop family (the
GGAA, GAGA, GCAA, and GAAA loops) that should stabilize base pairing in
the adjacent stem (37). Helices 2, 3, 4, and 5-1, in which nucleotides
58 and 67 and 14 and 262 are linked, should be found in sc73. New 5'
and 3' sites were created at nucleotide positions 249 and 80, respectively. sc73* RNA contained the same number of helices as sc73,
except that the sequence at nucleotides 24-28 (5'-AGCGG-3') of wild
type scRNA was changed to UGCC. In consequence, sc73* should lack the
characteristic tertiary interaction. In addition, sc51 contained two
subdomains of helix 5 (helix 5 was divided into three subdomains based
on the secondary structure as shown in Fig. 2). In sc51, nucleotides 92 and 238 were also linked with the sequence GGAA. Plasmids pTUE1331
(sc103), 1332 (sc73), 1333 (sc73*), and 1334 (sc51) were constructed by
annealing two complementary oligonucleotides corresponding to the
respective sense and antisense structures of the gene for each RNA. To
clone annealed fragments into pSP64, synthetic oligonucleotides were
designed to create HindIII and BamHI sites at the
5' and 3' end, respectively.
Plasmids pTUE1335 and pTUE1336 were constructed for in vitro
transcription of hammerhead ribozyme and B. subtilis tRNA
(AGC). These RNAs are thought to have double-stranded structures and were used in the gel retardation assay as competitors. The two annealed
oligonucleotides encoding each RNA were cloned into the HindIII-BamHI sites of pSP64.
The entire coding region of the B. subtilis HBsu gene
(hbs) was amplified by the polymerase chain reaction using
synthetic oligonucleotide primers and the genomic DNA of B. subtilis 168. Synthetic oligonucleotide A
(5'-GGCCCCATGGACAAAACAGAACTTATCAATGCG-3') containing a novel
NcoI site was used as one primer, and synthetic oligonucleotide B (5'-GCAGAGATCTTTTTCCGGCAACTGCGTCTTTAAGC-3') containing a novel BglII site was used as the other
primer. The resulting PCR fragment was cut with NcoI and
BglII and then inserted into the
NcoI-BglII sites of pQE60. This plasmid was
designated pTUE1337.
The 5' part of the B. subtilis hbs gene encoding the +1 to
+29 amino acids of HBsu was amplified by PCR using the synthetic oligonucleotide primers C and D (C,
5'-GAATGTAAAGCTTGGGAGGAGGTGAAAGGC-3'; D,
5'-GGGGGGTCTAGAAACAGAGTCAACTGC-3') and genomic DNA as a template. The 106-bp DNA product was cut with SmaI-XbaI and
inserted into the SmaI-XbaI sites of pDH88. The
resulting plasmid with the ribosome binding sequence and the truncated
gene positioned downstream of inducible promoter spac-1 was
designated pTUE1338 and used to construct B. subtilis UT1681.
The 3' part of the B. subtilis
ffh(his)6 gene encoding amino acids +1 to
+446 of B. subtilis Ffh and six histidine residues was amplified by PCR using the synthetic oligonucleotide primers E and
F (E, 5'-GAAATCGCGAATCCGG-3'; F, 5'-TATCACCAGCTCACCG-3'), and
pTUE820, which allows the overexpression of
ffh(his)6, was used as a template
(25). The PCR product was purified and cut with EcoRI and
then inserted into the EcoRI site of pDH88. The resulting
plasmid, designated pTUE1339, was used to construct B. subtilis strain UT1682.
The constructs were confirmed by physical mapping and DNA sequencing.
In Vitro Synthesis of RNAs--
Probe RNAs were transcribed
from BamHI-linearized plasmids using 35 units of SP6 RNA
polymerase (Takara Shuzo Ltd.) in the presence of
[ Cell Extract Preparation--
Cell lysates were prepared from
B. subtilis 168 culture that reached an absorbance reading
of 0.5 at 660 nm as described previously (38), except that the
suspensions were incubated in Buffer A (20 mM Tris-HCl, pH
7.8, 20 mM NH4Cl, 10 mM
(CH3COO)2Mg, and 5 mM
2-mercaptoethanol) containing 10 mg/ml lysozyme at 37 °C for 1 h before sonic oscillation. To purify the 10-kDa protein, cell extracts
were fractionated by ammonium sulfate saturation (65-90%). After
centrifugation at 50,000 × g for 20 min, precipitates
were resuspended in Buffer B (20 mM Tris-HCl, pH 7.8, 10 mM (CH3COO)2Mg, 5 mM
2-mercaptoethanol, and 0.25 M sucrose) and stored at
Protein Sequence--
Peptide sequences were obtained from
proteins separated by SDS-PAGE and blotted onto polyvinylidene
difluoride membranes. Blotted proteins were located by staining with
Coomassie Blue G (Sigma), excised, and examined using an Applied
Biosystems model 494 protein sequencer.
Construction of Strains--
To determine the functions of HBsu
in cell growth and protein secretion, plasmid pTUE1338 was introduced
into the B. subtilis 168 chromosome by competent cell
transformation (39). In the resulting strain, UT1681, the intact
hbs gene was under the control of the inducible
spac-1 promoter, whereas the truncated gene was controlled
by the intact promoter. Throughout the experiments, cells were cultured
in the presence of 2 mM IPTG containing solid or liquid medium.
To investigate the presence of complexes containing Ffh and HBsu
in vivo, B. subtilis strain UT1682 was
constructed by transferring plasmid pTUE1339 into the chromosome of
B. subtilis 168. In the resulting strain,
Ffh(His)6 was constitutively expressed. Replacement of the
desired genes was verified by PCR and/or Southern blotting.
Sucrose Gradient Centrifugation--
Cell extracts for sucrose
gradient sedimentation were prepared as described above for the
purification of the 10-kDa protein, except that the buffer contained
200 mM KOAc and 2 mM vanadyl ribonucleoside
complex. Extracts were layered onto 5-15% sucrose gradients and
separated by centrifugation for 15 h at 40,000 × g in a Beckman SW40 rotor at 4 °C, after which fractions
were manually collected from the top of the gradient. Aliquots from each fraction were prepared for RNA extraction or SDS-PAGE.
Production and Purification of Recombinant HBsu with a Tag
Consisting of Six Consecutive Histidine Residues at the Carboxyl
Terminus--
Recombinant HBsu tagged with six consecutive histidine
residues at the carboxyl-terminal was prepared from E. coli
M15 (pREP4) (Qiagen) harboring pTUE1337 by the same method as described
previously (27). The estimated purity of the protein was over 90%,
based on laser densitometry of the Coomassie Brilliant Blue-stained gel
using a Bio Image Analyzer (Millipore/Millige).
Gel Mobility Shift Assay--
RNA probes labeled with
32P (1 × 104 cpm) were incubated with
various amounts of purified proteins or B. subtilis cell
extracts in 10-µl reaction mixtures containing 14 mM
HEPES, pH 8, 45 mM KCl, 6 mM MgCl2,
1 mM dithiothreitol, 0.5 mM
phenylmethlysulfonyl fluoride, 1 µg of poly(dI-dC), and 0.3%
vanadium ribonucleoside complex. After a 30-min incubation at 25 °C,
samples were loaded onto a 4% nondenaturing polyacrylamide gel
containing 45 mM Tris-HCl, pH 8.3, 1 mM EDTA,
45 mM boric acid, and 2.5% glycerol. Dried gels were
exposed to x-ray films (Fujifilm Co.) at Northwestern Blotting, Western Blotting, and
Immunoprecipitation--
Proteins were separated by SDS-PAGE and then
transferred to polyvinylidene difluoride membranes by electroblotting.
For Northwestern analysis, membrane-bound proteins were renatured by
incubation in Buffer C (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 0.02% Ficoll (Type400;
Pharmacia Biotech Inc.), 0.02% polyvinylpyrrolidone, and 0.02% bovine
serum albumin). Membranes were blocked with a 2% solution of skim milk
in Buffer C and then rinsed with Buffer C containing 200 mM
KCl. For probing with radiolabeled RNA, membranes were incubated for
1 h with 32P-labeled RNA (5-10 × 104 Cerenkov cpm/ml) in Buffer C containing 200 mM KCl and 0.1% Triton X-100. After washing with Buffer C
containing 200 mM KCl and 0.01% Triton X-100, the
membranes were air-dried and exposed to film with enhancer screens for
autography or imaged using a BAS2000 Fuji image analyzer. For
immunoblots, membranes were blocked with 5% skim milk in Tris-buffered
saline (20 mM Tris-HCl (pH 7.6) and 150 mM
NaCl), probed with primary antiserum (1:1,000 dilution in 5% bovine
serum albumin/Tris-buffered saline) for 1 h, rinsed with
Tris-buffered saline containing 2% Triton X-100, and then incubated
with secondary antibody for 1 h. After washing with Tris-buffered
saline containing 2% Triton X-100, bound antibody was detected by
enhanced chemiluminescence. For immunoprecipitation, cell extracts (100 µl) were diluted at 4 °C in 8 volumes of immunoprecipitation buffer (50 mM Tris-HCl, pH 8, 0.1 mM EDTA,
Mg(OAc)2, 0.5 mM phenylmethylsulfonyl fluoride,
and 1 mM dithiothreitol) and mixed at 4 °C for 1 h
with 10 µl of antiserum and then mixed for 2 h with 100 ml of
protein A-Sepharose resin. The beads were pelleted by a brief
centrifugation, washed three times with immunoprecipitation buffer
containing 0.05% Nikkol, and then processed for protein or RNA
analysis. Antiserum against B. subtilis HBsu was kindly
provided by M. A. Marahiel.
Identification of a Protein That Binds to the scRNA Alu Domain in
B. subtilis Cell Lysates--
Fig. 1
shows that Ffh and scRNA co-sedimented on sucrose density gradients
with a sedimentation coefficient of 8 S, which is greater than would be
expected for a particle containing only Ffh and scRNA (data not shown).
Thus, the B. subtilis complex probably contains another
protein component(s). Moreover, the protein component(s), if present,
could bind the Alu domain consisting of helices 1-5 because
Ffh binds the S domain comprising helix 8. To identify the
Alu domain-binding protein(s) in B. subtilis cell
extract, we performed a RNA mobility shift assay. B. subtilis cell extracts were incubated with 32P-labeled
sc103 (Fig. 2B) containing
helices 1-5 synthesized in vitro. One distinct band
appeared, and the amount of complex formed was dependent on the amount
of protein added (Fig. 3, lanes
2-5). The mobility shift band corresponding to this band was also
obtained when full-length mature scRNA was used as a probe (data not
shown). This band was sensitive to Pronase K (data not shown),
indicating that B. subtilis cells express a protein that
binds to the scRNA Alu domain. In addition to sc103, we
designed three substrates to determine which helices would serve as
binding sites for this protein. We constructed two mutants that were
deletions of helix 5. We divided helix 5 into three subdomains
(referred to as helices 5-1, 5-2, and 5-3; Fig. 2). In addition to
helices 1 and 2, helix 5-3 and part of helix 5-2 were also deleted from
sc73 and sc73* substrates. In sc73*, five nucleotides (AGCGG) in the
loop connected to helix 3 were changed to UCGCC. As a result,
nucleotide complementation between the two loops connected to helices 3 and 4 was disrupted to diminish the pseudoknot structure between them
(Fig. 2). Mutant sc51 was composed of two subdivisions of helix 5 (helix 5-1 and 5-2), corresponding to domain II. Of these three
mutants, only sc73 formed the same amount of binding complexes as sc103
(Fig. 3, lanes 1-10), whereas sc51 had no activity (Fig. 3,
lanes 16-20). The sc73* mutant showed a significant band,
but the efficiency was reduced to 20% of that of sc103 (Fig. 3,
lanes 11-15).
Purification of the 10-kDa Protein--
A B. subtilis
cell lysate was fractionated with saturated ammonium sulfate
(65-90%), and the pellet was loaded onto a DEAE-Sepharose CL-6B
column (Pharmacia). As shown in Fig.
4A, a shift band with the same
mobility as that shown in Fig. 3 was found in fractions 6-12
(C2). Moreover, in fractions 7 and 8, more mobility shift band (C1) was obtained. This band, designated C1, may be
caused by the binding of homodimeric protein to the radiolabeled RNA probe, because this band disappeared and only band C2 was obtained when
a smaller volume of fraction 7 and 8 was used (data not shown). To
identify which protein in the fractions is responsible for Alu domain binding activity, fractions containing activity
from the DEAE-Sepharose CL-6B column (Fig. 4A, fractions
5-10) were individually concentrated to <50 µl and separated on a
denaturing SDS gel. The separated proteins were blotted onto a
polyvinylidene difluoride membrane and hybridized with
32P-labeled sc103 transcribed in vitro. After
washing the membrane to remove excess probe, a 10-kDa protein
interacted with the probe, and the amount of the positive band peaked
at fraction 7 (Fig. 4B). When aliquots of these fractions
were resolved by SDS-PAGE, the gel stained with Coomassie Blue
demonstrated that a single predominant protein band with an apparent
molecular weight of about 10,000 was responsible for the positive band
in the Northwestern blot (Fig. 4, B and C). The
protein was blotted onto a polyvinylidene difluoride membrane and
excised, and the amino acids were sequenced.
The 10-kDa Protein Is HBsu--
At the amino-terminal region of
the 10-kDa protein, the only sequence obtained was
MNKTELINAVAEASELSKKD. A search of the nonredundant protein database
using the BLAST search program revealed that this 10-kDa protein is
identical to histone-like protein (HBsu) (SwissProtTM
accession number P08821) (40). The identity of this 10-kDa protein as
HBsu was confirmed by immunoblotting using rabbit anti-B. subtilis HBsu antiserum (data not shown). However, we could not exclude the possibility that a low level of contaminating protein in
the fraction was responsible for the band shift. To confirm that HBsu
is identical to the 10-kDa protein, we expressed His-tagged HBsu in
E. coli and purified it using a Ni-NTA+ column.
The binding activity of the purified HBsu was tested by gel mobility
shift assay. A complex was formed with 32P-labeled sc73
(Fig. 5A, lane 1). To examine
binding specificity, three relevant RNAs (~100 nucleotides long) were
synthesized in vitro and used as competitors in the
(His)6-tagged HBsu-sc73 binding assay. RBZ indicates the
hammerhead ribozyme. Studies predict that RBZ and tRNAAla
contain double-stranded stem-loops, whereas the poly(A)+
competitor consists of 63 consecutive adenines and has no possible secondary structure. Among the competitors we constructed, only sc73
itself effectively competed for binding of the HBsu protein (Fig.
5A, lanes 2-4). The other mutants had no effect on the
binding (Fig. 5, B-D). These results confirmed that
B. subtilis HBsu protein specifically binds to the scRNA
Alu domain.
In Vivo Complex Formation of HBsu with Ffh via scRNA--
Data
from our in vitro studies indicate that HBsu can form a
complex with scRNA in cells. To determine whether or not HBsu interacts
directly with scRNA in vivo, we immunoprecipitated B. subtilis cell extracts with anti-HBsu antiserum and determined the
presence of scRNA within the HBsu immune complex by Northern blotting
with the 32P-labeled DNA fragment encoding mature scRNA.
scRNA was immunoprecipitated with the anti-HBsu antiserum but not with
rabbit preimmune antiserum (Fig.
6A, lanes 2 and 3),
demonstrating that B. subtilis scRNA associates with HBsu
in vivo. Because scRNA is known to be associated with
another protein, Ffh, it is more likely that Ffh-scRNA-HBsu forms a
ternary complex. To examine this notion, we constructed the B. subtilis mutant UT1682 in which chromosomally encoded Ffh is
tagged with six consecutive histidine residues at the carboxyl terminus
(Fig. 6B). A cell lysate of this mutant was mixed with Ni-NTA+ resin, and then a complex containing His-tagged Ffh
was precipitated. The complex was washed, and then we examined whether
or not scRNA and HBsu were present. Fig. 6C shows that both
HBsu and scRNA were complexed with
Ffh(His)6-Ni-NTA+ resin. Moreover, the amount
of HBsu diminished when lysates were incubated with ribonuclease A and
T1 before precipitation (Fig. 6C, lane 5). These data
suggest that HBsu-scRNA-Ffh form a complex in vivo and that
scRNA serves as a functional unit (backbone) for ternary complex
formation. We have recently shown that B. subtilis scRNA can
bind elongation factor G (38). However, the complex with Ffh
(His)6-NTA+ resin did not contain elongation
factor G (data not shown).
HBsu Is Required for Cell Growth--
To understand the function
of HBsu, we constructed plasmid pTUE1338, which would allow replacement
of the native HBsu with the IPTG-inducible spac-1 promoter.
In the resulting transformed strain, UT1681, the gene expression of
HBsu is regulated by IPTG. The transformants were able to grow as well
as wild type cells in the presence of IPTG (Fig.
7). However, if this transformant is
inoculated in the absence of IPTG, the growth is greatly inhibited, indicating that HBsu is essential for normal growth. These results are
consistent with those of Micka and Marahiel (41).
To date, only Ffh has been identified as a protein component of a
SRP-like particle in bacteria. Homologues of SRP9/SRP14 that can
interact with the Alu domain of SRP RNA have not yet been
identified. The present study demonstrated that the B. subtilis histone-like protein, HBsu, is an integral component of a
ribonucleoprotein complex containing at least Ffh and scRNA and that
HBsu can recognize the predicted secondary structure of the
Alu domain. HBsu of B. subtilis belongs to a
widespread family of histone-like proteins in bacteria (40, 42, 43),
and its function is essential for cell growth (41). This family
represents a group of small, basic, and abundant proteins that bind DNA
(44). Histone-like proteins play a role in DNA condensation. Like HU,
HBsu binds DNA. In contrast to HU, which predominantly forms
heterodimers, HBsu binds DNA as a homodimer (45). Because the function
of HBsu in the SRP-like particle remains to be resolved, several arguments favor the notion that HBsu is a true component of the B. subtilis riboprotein complex. (i) The immunoprecipitation
of cell lysates using anti-HBsu antiserum yielded scRNA (Fig.
6A). Moreover, immunoprecipitation identified only scRNA as
the RNA species in the immunoprecipitates (data not shown). (ii) Fig. 6C shows that HBsu co-precipitation with
(His)6-tagged Ffh depended on the presence of scRNA,
suggesting that scRNA serves as a functional unit (backbone) in this
complex. (iii) After fractionation on Although the mechanism of the interaction of B. subtilis
HBsu with the scRNA Alu domain was unknown, the crystal
structure of the monomeric and homodimeric B. stearothermophilus DNA-binding protein HU (BstHU) relative to the
mesophilic homologue B. subtilis HBsu was determined at a
resolution of 3 Å, and a model of nucleic acid-HU interaction was
proposed (45). Based on the proposed model, the secondary structure of
the BstHU monomer is represented as
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit of SRP receptor, respectively, have been identified
(22-25). Like SRP54, Ffh can be cross-linked to a variety of
functional signal sequences derived from both Gram-negative and
-positive bacteria (26, 27). Moreover, we demonstrated that the
depletion of SRP components or SRP receptor causes defects in
preprotein translocation (23, 24, 28).
-lactamase translocation (23). These effects can be compensated by
introducing human or E. coli SRP RNAs, indicating that the
essential function of SRP RNAs is evolutionarily conserved and that it
locates in the region of helix 8. The structural features of SRP RNA
and eubacterial phylogeny based upon the 16S rRNA sequence reveal a
discrepancy (21). Thermus thermophilus has been placed in the earliest branches of the tree, whereas bacilli are thought to have
arisen considerably later in eubacterial evolution. The question then
arose as to the advantages conferred upon Bacillus and other
Gram-positive bacteria by the preservation of helices 1-5 if
eukaryotic RNAs represent the prototype structure. To identify the
structural requirements for B. subtilis scRNA, we
constructed mutants in which individual helices were deleted and
assayed their importance in vivo. The results showed that
helices 1-4 and part of helix 5 are not essential for vegetative
growth but are needed for the formation of heat-resistant spores (35).
Moreover, we propose that scRNA combined with Ffh to form a SRP-like
particle functions in the translocation of sporulation-specific
proteins such as penicillin-binding protein 5* to the interspace
between the mother cell and the prespore. To understand the function of the Alu domain of B. subtilis scRNA, it is first
necessary to identify protein(s) that can bind this region.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Plasmids and bacterial strains
-32P]CTP (400 Ci/mmol; Amersham International) using
the Riboprobe Gemini system II Buffers Kit (Promega). The sample was
precipitated twice by ethanol, and then purified RNAs were resolved in
sterile deionized water. The concentrations of radiolabeled RNAs were determined from the specific activity of the [
-32P]CTP
incorporated into the transcripts. Before use, RNAs were renatured by
incubating them for 15 min at 65 °C, followed by a slow cooling to
room temperature. Competitor RNAs were also transcribed from
BamHI-linearized plasmids as described above, except that
nonlabeled CTP was used instead of [
-32P]CTP.
20 °C until use.
80 °C, typically for
16 h.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Ffh and scRNA co-sediment in sucrose density
gradient. Crude cell extracts were layered over a 5-15% sucrose
gradient and centrifuged for 10 h at 40,000 × g.
The gradient fractions were collected and divided into two aliquots.
One aliquot was analyzed by Western blot using an antibody against Ffh
(A), and total RNA from the other aliquot was analyzed by
Northern blot using a scRNA-specific probe (B).
Sedimentation values of the protein standards (cytochrome oxidase, 5.8 S; fibrinogen, 8.0 S; catalase, 11 S) are indicated.
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Fig. 2.
Predicted secondary structures of wild type
and mutant scRNAs used as probes. A, schematic
representation of the secondary structures of wild type scRNA. The
predicted secondary structures were generated using the RNA structure
editing computer program DNASIS. Helices 1-8 were numbered according
to the structure of human SRP RNA (7SL RNA) proposed by Larsen and
Zwieb (29). We divided helix 5 into three subdomains (5-1, 5-2, and
5-3), based on the structure isolated at the bulge. The outlined
letters (helices 6 and 7) indicate the helices deleted from scRNA
compared with human SRP RNA. B, sequences and possible
secondary structures of the mutant scRNAs used as probes in gel
mobility shift assays. Nucleotides were numbered according to wild type
scRNA. The two sequences of five nucleotides boxed in sc103
and sc73 are complementary and should maintain a pseudoknot structure
between two loops. Asterisks (*) indicate nucleotides
changed in sc73*. A possible G-U base pairing is indicated ( ).
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Fig. 3.
A gel mobility shift of scRNA by B. subtilis cytoplasmic proteins. 32P-labeled
mutant scRNA, sc103 (lanes 1-5), sc73 (lanes
6-10), sc73* (lanes 11-15), and sc51 (lanes
16-20) were incubated with 0 (lanes 1, 6, 11, and
16), 0.5 (lanes 2, 7, 12, and 17), 1.5 (lanes 3, 8, 13, and 18), 10 (lanes 4, 9, 14, and 19), and 30 (lanes 5, 10, 15, and
20) µg of protein prepared from B. subtilis 168 as described under "Experimental Procedures." C and
FP indicate RNA-protein mobility shift complex and
protein-free RNA probes, respectively.
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Fig. 4.
Purification of the 10-kDa protein from
B. subtilis cell extract. The 65-90% ammonium
sulfate precipitate of the cell extract was loaded onto a Sepharose
CL-6B column. The proteins were eluted with 0.25 M NaOAc,
followed by 2 M NaOAc. The fractionated proteins were
assayed by the gel mobility shift assay (A) and by
Northwestern blotting (B). C, Sepharose CL-6B
fractions were concentrated, separated by denaturing SDS-PAGE, and
stained with Coomassie Blue, and then the proteins were sequenced.
C1 and C2 indicate the position of RNA-protein
complex. FP indicates a protein-free RNA probe.
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Fig. 5.
Binding specificity of purified HBsu tagged
with six histidines at the carboxyl-terminal. The ability of
purified HBsu to bind [ -32P]CTP-labeled sc73 in the
presence of competitor RNAs was assayed by gel mobility shift assay.
Competing RNAs were sc73 itself (A), hammerhead ribozyme
(B), 60 consecutive adenines (poly A;
C), and tRNA(AGC) (D). In A-D, the first
lanes (lanes 1, 5, 9, and 13) show binding in the
absence of competitor; all lanes contain 1 µg of yeast RNA as a
nonspecific competitor. Each unlabeled, specific competitor is
presented in three sets with the first lanes (lanes 2, 6, 10, and 14) containing a 10-fold molar excess of the
indicated competitor, the second lanes (lanes 3, 7, 11, and
15) containing a 20-fold molar excess of the indicated
competitor, and the third lanes (lanes 4, 8, 12, and
16) containing a 100-fold molar excess of the indicated
competitor. The mobility of the free probe (FP) and shifted
complex (C) on the 4% nondenaturing gel are
indicated.
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Fig. 6.
HBsu interacts with Ffh and scRNA to form a
ternary complex in vivo. A, HBsu interacts with
intracellular scRNA. Cell extracts were prepared from B. subtilis 168 and immunoprecipitated with a polyclonal antibody
against HBsu (IP Ab, immunoprecipitation antibody)
(lane 2) or with preimmune antiserum (lane 3).
Co-precipitated scRNA was extracted using the same volume of phenol and
analyzed by Northern blot to detect scRNA. The mobility of the scRNA
was estimated from that of purified scRNA on the same gel (lane
1). B, Campbell-like integration of plasmid pTUE1339
containing ffh(his)6 fusion into the
chromosome of wild type B. subtilis 168. In the resulting
strain, UT1682, ffh(his)6 fusion is
under the control of the native ffh promoter in front of the
upstream gene (ylxM) (24). C, ternary complex
formation among Ffh, scRNA, and HBsu in vivo. Cell extracts
were prepared from B. subtilis strain UT1682, and
Ffh(His)6 was precipitated with the Ni-NTA+
resin. Proteins in the immunocomplex were detected using an antibody
against Ffh itself (lanes 1 and 2) and HBsu
(lanes 5 and 6), respectively. Co-precipitated
scRNA (lanes 3 and 4) was detected by Northern
hybridization. Before chromatography on Ni-NTA+-agarose,
samples were incubated with (lanes 1, 3, and 5)
or without (lanes 2, 4, and 6) 2 mg of RNase A
and T1.
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Fig. 7.
Growth curves during the depletion of HBsu.
B. subtilis strain UT1681 was grown in the presence ( ) or
absence (
) of 2 mM IPTG.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amino pentyl-agarose and
DEAE-Sepharose CL-6B that had been used to purify mammalian SRP, scRNA,
Ffh, and HBsu were still co-eluted by a relatively high salt
concentration (250 mM KOAc acetate),2 suggesting that
the three constituents formed a complex in a salt-resistant manner.
1-
2-
1-
2-
3-
3 with
two distinct halves (Fig. 8A).
The amino-terminal half consists of two
-helices connected by a
broad turn (
1) to create a V-shaped supersecondary structure. The
carboxyl-terminal half consists mainly of a three-stranded antiparallel
-sheet that spans the top of the V formed by the two helices. The
two halves of the structure are connected by a
-turn between
2
and strand 1, which has a highly conserved glycine residue. Fig.
8B shows that the amino acid residues among B. subtilis HBsu, YonN, and B. stearothermophilus HU are
highly conserved (more than 90% amino acid sequence homology).
Therefore, a similar supersecondary structure could be drawn for
B. subtilis HBsu. Two monomers wrap around each other to
produce a novel dimeric structure. The base of the molecule is formed
by the two V-shaped helical supersecondary structures packed such that
the two
2 helices are in contact roughly at a right angle, and the
two
1 helices are on either side. Moreover, the visible parts of the
arms, together with the two symmetry-related strand 3 between them,
create a concave surface on the dimer. The helical depression has a
diameter of 25 Å, indicating that the protein electrostatically
interacts with the DNA. A mammalian heterodimer consisting of 9- and
14-kDa polypeptides (SRP9/SRP14) folds with a strikingly similar
concave structure, regardless of the low amino acid sequence similarity
among B. subtilis HBsu, SRP9, and SRP14. Birse et
al. (36) showed that SRP9 and SRP14 are structurally homologous,
containing the same
-
-
-
-
fold. According to their model,
the heterodimer has a pseudo 2-fold symmetry and is saddle-like,
comprising a rigidly curved six-stranded amphipathic
-sheet with the
four helices packed on the convex side, and the exposed concave surface
being lined with positively charged residues. The SRP9/SRP14
heterodimer may be the latest member of a growing family of small
/
RNA-binding proteins. These striking structural similarities
indicate that saddle-like folding in the HBsu homodimer is important
for scRNA Alu domain binding and that curvature of the HBsu
-sheet is suitable for the tertiary structure of the RNA maintained
by the five base pairings between the two loops. However, we have not
examined whether or not HBsu dimerization is required for binding.
Nonetheless, mutational analysis supported this notion because
nucleotide changes diminishing the formation of this tertiary
interaction notably reduced the ability of HBsu to bind the scRNA
Alu domain (Fig. 3, sc73*). The dsRNA binding
consensus sequence (KGFG-F-V-F) conserved among E. coli
RNaseIII, human TAR-binding protein, and Drosophila staufen
protein was found between
strands 1 and 2 in HBsu (Fig. 8)
(46).
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Fig. 8.
Structure-based amino acid sequence alignment
comparing histone-like proteins from Gram-positive and -negative
bacteria. A, schematic representation of the tertiary
structure of B. stearothermophilus DNA-binding protein II
dimer. The protein model was generated by Molscript using
crystallographic data from Tanaka et al. (45). The protein
backbone is depicted by ribbons. B, amino acid sequence
alignment of bacterial histone-like proteins. HBsu B. sub.,
B. subtilis HBsu; YonN B. sub., B. subtilis YonN; HBS B. ste., B. stearothermophilus DNA-binding protein II; NS-1 E. coli, E. coli NS-1 (HU ); NS-2 E. coli,
E. coli NS-2 (HU
). The secondary structure distribution
is based on an electron density map of B. stearothermophilus
DNA-binding protein II (45) and is represented by
-strands
(horizontal black bars) and
-helices (horizontal
gray bars). Consensus of at least 50% identical charged residues
is denoted by
; residues contributing to the hydrophobic core are
depicted by
.
The present results raise the question of the significance of the
interaction between HBsu and its RNA ligand. We demonstrated that
physiological defects of the depletion of scRNA can be compensated by
E. coli 4.5 S RNA that completely lacks the Alu
domain (23). Moreover, depleting scRNA of its Alu domain did
not affect vegetative growth. Therefore, the essential nature of HBsu
protein would appear to be unrelated to its role in B. subtilis SRP function. However, we also showed that the growth
rates of the scRNA mutant without the Alu domain are
inhibited at the end of the vegetative phase (35). The frequency of
heat-resistant spores was concomitantly reduced to 20% of the level in
the wild type. Secretory proteins, including -amylase and alkaline
protease in B. subtilis, are synthesized at the end of
vegetative growth and actively secreted into the culture medium.
Moreover, during sporulation, many proteins are synthesized in the
mother cell, translocated across the membrane, and localized in the
interspace between the mother cell and the forespore. The
penicillin-binding protein PBP5* is representative of proteins located
in the outer forespore membrane. This is specifically synthesized in
the mother cell at the late stage of sporulation and is required for
the heat-resistant spore formation. Bunai et al. (26, 47)
demonstrated that Ffh protein that is a component of B. subtilis SRP can bind the signal sequence of PBP5*. Based on these
data, B. subtilis SRP function appears to be required for
translocation during the late stage of vegetative growth and during
sporulation. The loss of function that arises when the Alu
domain is deleted suggests that the Alu domain, perhaps
including HBsu, is required for B. subtilis SRP function. We
also demonstrated the requirement for the Alu domain by
showing that Clostridium perfringens scRNA that has this
domain can compensate for both vegetative growth and spore formation in
B. subtilis cells depleted of scRNA (48).
Brown et al. (49) demonstrated that the stable assembly of yeast SRP in vivo relies on the presence of all subunits. The levels of scR1 RNA (SRP RNA) and of other SRP proteins in yeast were significantly reduced in strains lacking any one oSRP14p, SRP21p, SRP68p, or SRP72p. In contrast to yeast, HBsu depletion caused defective growth of the UT1681 strain directly incubated in the absence of IPTG. Therefore, evaluating the function of Hbsu in the early stage of SRP biogenesis under these conditions is difficult.
Although several proteins may interact with SRP RNA, HBsu appears to be
the first one directly identified as a scRNA Alu
domain-binding protein in B. subtilis.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. I. Tanaka at Hokkaido University for providing the 3D protein model for B. stearothermophilus DNA-binding protein II. We are grateful to N. Foster for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by Grants-in Aid for Scientific Research on Priority Areas from the Ministry of Education, Sciences and Culture, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel./Fax:
81-298-53-4661; E-mail: nakamura.kouji{at}nifty.ne.jp.
2 K. Nakamura, S.-i. Yahagi, T. Yamazaki, and K. Yamane, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
SRP, signal
recognition particle;
bp, base pair(s);
IPTG, isopropyl-1-thio--galactopyranoside;
Ni-NTA+, Ni2+-nitrilotriacetic acid;
scRNA, small cytoplasmic RNA;
PAGE, polyacrylamide gel electrophoresis;
PCR, polymerase chain
reaction.
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REFERENCES |
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