From the Institute of Biological Sciences, University of Tsukuba, Tsukuba-shi, Ibaraki 305-8572, Japan and the § Department of Industrial Chemistry, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino-shi, Chiba 275-0016, Japan
Received for publication, February 13, 2001, and in revised form, April 5, 2001
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ABSTRACT |
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Escherichia coli cells contain abundant
amounts of metabolically stable 4.5 S RNA. Consisting of 114 nucleotides, 4.5 S RNA is structurally homologous to mammalian 7 S RNA,
and it plays an essential role in targeting proteins containing signal
peptide to the secretory apparatus by forming an signal
recognition-like particle with Ffh protein. It also binds
independently to protein elongation factor G (EF-G) and functions in
the translation process. This RNA contains a phylogenetically conserved
RNA domain, the predicted secondary structure of which consists of a
hairpin motif with two bulges. We examined the binding activity of
mutants with systematic deletions to define the minimal functional
interaction domain of 4.5 S RNA that interacts with EF-G. This domain
consisted of 35-nucleotides extending from 36 to 70 nucleotides of
mature 4.5 S RNA and contained two conserved bulges in which mutations of A47, A60, G61, C62, A63, and A67 diminished binding to EF-G, whereas
those at A39, C40, C41, A42, G48, and G49 did not affect binding. These
data suggested that the 10 nucleotides in 4.5 S RNA, which are
conserved between 4.5 S RNA and 23 S rRNA, have a key role for EF-G
binding. Based on the NMR-derived structure of mutant A47U, we further
verified that substituting U at A47 causes striking structural changes
and the loss of the symmetrical bulge. These results indicate the
mechanism by which EF-G interacts with 4.5 S RNA and the importance of
the bulge structure for EF-G binding.
Escherichia coli 4.5 S RNA is a structural homologue of
signal recognition particle
(SRP)1 7 S RNA in eukaryotic
cells (1). Larsen et al. (2) proposed that the 300 nucleotides of mammalian 7 S-like RNA contain 8 helices (numbered 1 to
8). Based on the predicted secondary structure, almost all 7 S-like
RNAs of eukaryotes and archaebacteria are about 300 nucleotides long
and contain 8 helices. In contrast to the structural integrity of these
7 S-like RNAs, eubacterial 7 S-like RNAs differ in size and have
relatively little sequence identity (29%) outside of the conserved
domain. However, eubacterial 7 S-like RNAs have an identical
22-nucleotide sequence within helix 8. A phylogenetic study has
revealed differences in the length and secondary structure of 7 S-like
RNAs between Gram-positive and Gram-negative bacteria. Almost all
Gram-negative bacteria, including E. coli and
Pseudomonas aeruginosa 7 S-like RNAs are around 120 nucleotides long and can be folded into a single hairpin, corresponding
to helix 8 of mammalian 7 S RNA. The 7 S-like RNAs of Gram-positive
bacteria, including Bacillus subtilis (3, 4) and
Clostridium perfringens (5) consist of ~270 nucleotides, and the predicted secondary stricture is strikingly similar to that
eukaryotic 7 S-like RNA, although they lack helix 7. In eubacterial cells, 7 S-like RNA and Ffh protein, which is a homologue of SRP54, constitute a ribonucleoprotein particle and are involved in protein secretion (6-8). On the other hand, we showed that 4.5 S RNA can bind
to the protein elongation factor, EF-G, indicating that 4.5 S RNA is
also involved in translation (9, 10). Indeed, 4.5 S RNA depletion
causes a significant loss of translation (11-13), and suppressors of
the 4.5 S RNA requirement reside in genes encoding components of the
translation system, such as EF-G, 23 S rRNA, and tRNA synthetases (14,
15). In addition, 4.5 S RNA depletion leads to an increase in the
amount of EF-G associated with ribosomes, suggesting that 4.5 S RNA is
concerned with the mode by which EF-G associates with ribosomes (16).
Therefore, we proposed that 4.5 S RNA is a bifunctional molecule that
functions in translation and protein secretion by binding each protein.
Because a ternary complex consisting of 4.5 S RNA, Ffh, and EF-G was
undetectable in electrophoretic mobility shift assays, the binding site
for both proteins overlaps in 4.5 S RNA. Wood et al. (17)
demonstrated that nucleotides within the single-stranded region in
helix 8 are important both for Ffh binding to the RNA and for optimal function of the RNA in vivo. This bulged structure is
phylogenetically conserved among the 7 S-like RNA family (18). In
contrast to accumulating information about Ffh binding, that structural
features of 4.5 S RNA that are recognized by EF-G have not been
investigated. The decanucleotide sequences from 1068 to 1077 of 23 S
RNA (5'-GAAGCAGCCA-3') and from 58-67 of mature 4.5 S RNA are
identical (9, 15, 16). The corresponding region of 23 S RNA is
considered important for one of the EF-G binding sites (19-21).
However, the decanucleotide alone is not sufficient for binding EF-G,
suggesting that more higher level of structure of conserved bulge is
also important for protein binding (9, 16). We investigated the RNA
sequence and secondary structures affecting EF-G binding as follows. We performed deletion analysis of the 4.5 S RNA sequences, systematically altered bases and secondary structures, and assessed the consequences of these changes in vitro and in vivo. An NMR
structural study of a mutant 4.5 S RNA (A47U) suggested that the bulged
structure is important for protein binding.
Bacterial Strains and Plasmids--
Escherichia coli
strain S1192 (HfrH relA1 spoT1lacIq
ffs::kan591 ( In Vitro Synthesis of 32P-labeled 4.5 S RNAs with or
without Mutation--
The DNA templates for in vitro
transcription were generated by BamHI digestion of plasmid
DNAs of the pSP64 series encoding wild-type or mutant 4.5 S RNA. The
transcription reaction contained 40 mM Tris-HCl (pH 7.9), 6 mM MgCl2, 10 mM dithiothreitol, 2 mM spermidine, 50 µg/µl bovine serum albumin, 10 mM NaCl, 0.5 mM each of ATP, UTP, GTP, 25 µM CTP, 70 units of RNase inhibitor (Takara Shuzo Co.,
Ltd., Kyoto, Japan), 10 µl of [ Production and Purification of Recombinant EF-G with a Tag
Consisting of Six Consecutive Histidine Residues at the Carboxyl
Terminus--
E. coli EF-G mutant (EF-G Nitrocellulose Filtration Assay--
The 32P-labeled
4.5 S RNA probes (0.15 nM, 1.3 × 1018
cpm/mol) were incubated for 1 min at room temperature with various
concentrations of purified EF-G (0.001-10 µM) in 10 µl
of buffer (20 mM Tris-HCl (pH 7.4), 10 mM
MgCl2, 50 mM NaCl, 0.3% vanadium
ribonucleoside complex, and 1 unit of poly (dI-dC)). The mixture was
then diluted with 0.5 ml of washing buffer containing 20 mM
Tris-HCl (pH 7.4), 10 mM MgCl2, 100 mM NaCl, and 10% glycerol and immediately passed through
nitrocellulose filters (Advantec, 0.45-nm pore size; Toyo Roshi Kaisha,
Tokyo, Japan), which were washed with 1.5 ml of washing buffer. The
levels of radioactivity remaining on the filters were determined using
a liquid scintillation counter (LS5000TA , Beckman).
RNA Synthesis and Purification for NMR Study--
The
49-nucleotide fragment corresponding to the region from 29 to 77 of
wild-type 4.5 S RNA (SRP49) was made by annealing two synthetic
oligonucleotides. The oligonucleotides were designed to attach the T7
phage promoter sequence at the 5' end of RNA coding region. The
49-nucleotide DNA fragment encoding a mutant 4.5 S RNA where A at
position 47 of wild-type 4.5 S RNA is replaced by U was also
constructed (A47U). The SRP49 and the mutant (A47U) were synthesized
using an Ampliscribe T7 High Yield Transcription Kit (Epicentre
Technologies Corporation). We prepared 15N-labeled RNA
using 15N-NTPs (99.0 atom %, Nippon Sanso, Japan). After
transcription, RNAs were purified by 15% polyacrylamide gel
electrophoresis under denaturing conditions with 7 M urea.
The RNAs were eluted from gel in 0.3 M sodium acetate,
precipitated with ethanol, and desalted by ultrafiltration using
Centricon-3 (exclusion molecular weight 3,000) (Amicon).
NMR Experiments--
The purified RNAs were incubated for 5 min
at 95 °C and then cooled on ice. Samples containing 0.2-0.6
mM RNA were dissolved in 200 µl of 10 mM
sodium phosphate buffer (pH 6.3) in 90% H2O, 10%
D2O, and NMR spectra were measured using a DRX-500
spectrometer (Bruker) at probe temperatures of 10-50 °C with
symmetrical NMR microtubes (Shigemi Co., Ltd., Tokyo, Japan).
Solvent signals were suppressed by the jump-and-return pulse (22) for
all experiments with pulse intervals of 65 µs. For one-dimensional
measurements, the spectral width was 25 ppm, data points of 32 K were
used, and the number of scans was 128. Prior to Fourier transformation, line broadening of 3 Hz was applied. Two-dimensional NOESY experiments (23) were acquired with a mixing time of 150 ms. 512 free
induction decays of 2 K data points were collected using the
States-TPPI (time proportional phase incrementation) methods (23); the
number of scans was 256. Prior to Fourier transformation, Binding Activity of 4.5 S RNA to EF-G--
E.
coli 4.5 S RNA can bind both Ffh and EF-G. We examined the dose
dependence of Ffh and EF-G Effects of Point Mutations in Conserved Bulge Structures on Protein
Binding--
Fig. 1 shows that E. coli 4.5 S RNA can be folded into a single hairpin. This structure
is considered homologous to domain IV of mammalian SRP RNA (7 SL RNA).
In addition to the secondary structure, several nucleotides are highly
conserved. These lie with the tetranucleotide loop and its
adjacent single-stranded bulged regions. No other regions are
universally conserved in the primary sequence of 7 SL-like RNAs.
Moreover, among bacterial SRP RNAs, the numbers and structures of
single-stranded bulges are conserved (25). A mutational study
demonstrated that nucleotides within the bulge regions are important
for Ffh binding to the RNA (26, 27). To determine the role of the
bulge(s) and to identify which nucleotides are essential for EF-G
binding, we performed site-directed mutagenesis studies on each bulge
region. The ability of EF-G to bind the mutant 4.5 S RNAs was examined using a nitrocellulose filtration assay (Fig.
2). Within bulge A, point mutations at
positions 47, 60, 61, 62, and 63 completely abolished binding, whereas
those at G48U and G49C did not. A mutation from A to U at position 67, which lies within bulge B, also completely abolished the ability to
bind EF-G, whereas point mutations within the opposed bulged residues
at positions 39, 40, 41, and 42 did not. In contrast, all point
mutations induced within bulges C, D, and E did not affect binding to
EF-G. These results suggest that the nucleotide region that
includes bulges A and B is necessary for EF-G binding.
Systematic Deletion of 4.5 S RNA to Produce the Minimal Binding
Site for EF-G--
As a prerequisite to structural studies of the RNA
required for protein binding, a minimal RNA site for specific protein
binding had to be developed. This was accomplished by the systematic
deletion of entire bulges or portions of helices from wild-type 4.5 S
RNA. Genes encoding deletions were constructed by annealing two
complementary single-stranded DNAs. Each deletion mutant contained a
complementary sequence at the 5' and 3' termini (5'-GGGGG-3' and
CCCCCACCC-3') to minimize the potential for alternative secondary
structure formation as a result of deletion (Fig.
3A). Mutants 1 and 2, in which bulges E or
both D and E were deleted, bound to EF-G with wild-type affinity (Fig.
3B). Another mutant with three deleted bulges (C, D, and E)
also bound to EF-G with wild-type affinity. Mutant 3 consisted of
bulges A and B in addition to the conserved domain IV, demonstrating
that the structure of mutant 3 contains all the elements required for
interaction with EF-G. To assess the importance of bulges A and B, we
constructed mutants 4 and 5 that contained either bulge A or B in
addition to the stem-loop structure. Fig. 3B shows that
bulge A by itself has and can maintain appreciable binding
affinity, whereas mutant 5 completely loss the activity. These results
indicate that bulge A constitutes a minimal site in 4.5 S RNA required
for binding to EF-G.
Function of the Deletion Mutant of 4.5 S RNA in Vivo--
To
examine the ability of the constructed deletion mutants shown in Fig. 3
to restore the growth of E. coli S1192 in the absence of
IPTG, wild-type and deletion mutant 4.5 S RNA genes were inserted into
plasmid pKK223-3 (-op) and introduced into S1192. The introduced genes
were under the control of the tac promoter with a deleted operator sequence in all constructs. Therefore, the expression of 4.5 S
RNA on the plasmid was independent of IPTG. The single copy of the 4.5 S RNA gene in the chromosome of S1192 is regulated by the
lac repressor and requires IPTG for growth except when harboring a plasmid that provides 4.5 S RNA function. Therefore, growth
in the absence of IPTG would be a good indicator of mutant function.
The growth was maintained by a strain harboring wild-type 4.5 S RNA
(Fig. 4) but was abolished by a plasmid
without an insert in the absence of IPTG (data not shown), indicating
that the phenotype of plasmid-borne 4.5 S RNA can be assessed using
this in vivo system. The growth of transformants expressing
mutants 1 and 2 was identical to that of wild-type 4.5 S RNA (Fig. 4).
In contrast, mutants 4 and 5 no longer supported the growth of S1192
(Fig. 4). Mutant 3, which has decreased binding activity to EF-G, can restore the growth of S1192 but to a level of only half that of wild-type 4.5 S RNA. These results indicate a close relationship between the protein binding and growth restoration activity of 4.5 S
RNA-depleted cells.
Comparison of NMR Spectra of SRP49 and A47U Mutant--
Fig.
5a shows two-dimensional
15N-1H HMQC spectra (imino proton region) of
the SRP49 (black) and A47U (red) at 25 °C. The
imino proton resonances of G and U were distinguished by their
corresponding 15N chemical shifts. The resonances were
assigned by analyzing two-dimensional NOESY spectra as follows (data
not shown). The imino proton resonances of three SRP49 G:U pairs
U36:G70, U37:G39, and U45:G64 were identified by their unique chemical
shifts and strong intra-base pair NOEs. The imino proton resonance of
G53 in the GGAA tetraloop was also identified by its unique chemical
shift (25, 28). Starting from these unique resonances,
resonances were sequentially assigned by NOE connectivity as
U36/G70-G5-G4-G3-G2-G1, U45/G64-G44, and G53-G57-G58. The assignments
of imino protons of SRP49 in this study were consistent with earlier
NMR findings of SRP RNA (25, 28, 29). The imino proton resonances of
U37, U38, G43, G49, U50, G54, G61, and G69 could not be observed or
assigned because of broadening of the resonances, suggesting that these
imino protons are not involved in hydrogen bonding. These results are
summarized as the secondary structure shown in Fig. 5b. The
spectra of SRP49 and A47U are almost identical except for three
resonances (13.54, 13.25, and 13.02 ppm, Fig. 5a, red
triangle) and the disappearance of the two resonances (11.85 and
11.34 ppm, Fig. 5a, black triangle). From the analysis of
two-dimensional NOESY spectra, most of the imino proton resonances were
assigned as described above (data not shown). The two missing
resonances were assigned to a U45:G64 pair, because this pair is
closest to the substituted residue (A47U) among the observed G:U pairs.
This assignment was consistent with the fact that the resonances at
11.90 and 11.43 ppm were assigned to the U36:G70 pair based on the NOE
connectivity of U36/G70-G5-G4-G3-G2-G1. Although the new resonances
could not be assigned sequentially because of the lack of NOEs, the
resonance at 13.54 ppm was identified as an A:U pair because of the
presence of a typical NOE between the imino proton of U and the H2
proton of A (data not shown). Because the U45:G64 pair disintegrated and a new A:U pair formed in the A47U mutant, we proposed the secondary
structure shown in Fig. 5c, in which U45 bulges outward. The
proposed secondary structure is similar to that of SRP49 except for the
proposed EF-G binding region, which is consistent with the fact the
spectra of SRP49 and the A47U mutant were almost identical except for
five resonances. Accordingly, we suggest that the conformation around
U47 in the A47U mutant differed from that of the corresponding region
in SRP49 and that other regions were similar between the A47U mutant
and SRP49. Thus, the loss of EF-G binding activity in A47U may result
from destruction of the active conformation around A47.
Our previous study suggested that 4.5 S RNA is dual function and
participates in both translation and secretion by interacting with EF-G
and Ffh, respectively (9, 16). Here we examined the region required for
EF-G binding by introducing substitutions and deletions with the 4.5 S
RNA coding region of the ffs gene. The effects of sequence
elements and the secondary structure of phylogenetically conserved
bulged regions were measured by mutagenic alteration of 4.5 S RNA.
Changing any of the bulges C, D, or E did not significantly affect the
binding activity. Moreover, even in bulge A or B, changes in nucleotide
positions 39, 40, 41, 42, 48, and 49 did not reveal a key role in EF-G
protein binding. In contrast, nucleotides 60, 61, 62, 63, and 64 in the
conserved decanucleotide sequence were important for efficient binding
because some mutations at this site almost completely abolished the
binding. This result is consistent with our data showing that wild-type 4.5 S RNA can compete with 23 S RNA for binding to EF-G, but mutants with base substitutions in the decanucleotides cannot (9, 16). On the
other hand, Wood et al. (17) demonstrated that point mutations within the bulged residues at positions 47, 48, 49, 61, and
62 completely abolish binding to Ffh, whereas mutations at positions 60 and 63 do not affect the binding. Taken together, these data suggest
that nucleotides within single-stranded regions in bulges A and B are
important for binding to both proteins and that the nucleotides
essential for binding to each protein are overlapping but not
identical. Our present data using deletion mutants indicated that
mutant 2, consisting of 49 nucleotides (SRP49), is sufficient for
protein binding and can restore the growth of 4.5 S RNA conditional
mutant in the absence of IPTG. This 4.5 S RNA is the shortest among
known SRP 7 S-like RNAs. Defining regions for EF-G binding would be
useful in generating a minimal structure that would allow structural
analysis by NMR. The structures of 24-, 28-, and 43-nucleotide
fragments of 4.5 S RNA have been studied using multidimensional NMR
(25, 29, 30). The crystal structure of the complex between the
RNA-binding domain of Ffh (M domain) and the fragment of 4.5 S
RNA has recently been determined at a resolution of 1.8 Å (31). This
result demonstrates that in the symmetric loop (bulge A), stacking of
five consecutive non-canonical base pairs generates a unique helical
structure with a shallow minor groove. This groove in the symmetrical
internal loop A (bulge A, in this study) is recognized by the
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
imm434nin5
XhoI::
(Ptacffs)) was a gift from S. Brown (Department of Molecular Biology, University of Copenhagen,
Denmark) (14). The 5' portion of the normal chromosomal 4.5 S RNA gene
in this strain has been replaced by a kanamycin-resistance determinant. The chromosome contains a second bacterial ffs gene that is
expressed from the inducible Ptac promoter as a part of a
recombinant vector. A vector used to prepare the
32P-labeled RNA was pSP64 (Promega, Madison, WI). Plasmid
pKK223-3 (-op) is a derivative of pKK223 (Amersham Pharmacia Biotech,
Uppsala, Sweden), which contains the promoter sequence of the
tac promoter without the putative repressor-binding site
(16). Therefore, this plasmid can allow the gene cloned under the
tac promoter to constitutively express in E. coli
cells. The 4.5 S RNA genes with or without mutations were synthesized
by annealing two chemically synthetic oligo DNAs encoding each mutant
and introducing them between the BamHI/PstI sites
of pKK223-3 (-op) or between the BamHI/HindIII
sites of pSP64.
-32P]CTP (400 Ci/mmol, Amersham Pharmacia Biotech, International, Little Chalfont,
Buckinghamshire, UK), 1 pmol of linearized DNA fragment, and 35 units
of SP6 RNA polymerase (Takara Shuzo). After reaction, RNA was
precipitated with ethanol twice. Purified RNAs were resolved in sterile
and deionized water. The concentrations of radiolabeled RNAs were
determined from the specific activity of [
-32P]CTP
incorporation into the transcripts. Prior to use, RNAs were renatured
by incubation for 15 min at 65 °C followed by slow cooling to room temperature.
-(1-91)), with a
deletion of all upstream sequence up to and including the second
GTP-binding sequence element, was expressed and purified as described
by Suzuma et al. (10). To express EF-G
(1-91), E. coli M15 harboring both pREP4 (Qiagen, Chatsworth, CA), and the
plasmid encoding mutant EF-G was cultured on LB plates in the presence
of 50 µg/ml of ampicillin. A single colony was inoculated into 20 ml
of 2XTY medium containing 100 µg/ml ampicillin and 10 µg/ml
kanamycin and then incubated for 16 h at 37 °C. A 10-ml
inoculum of the overnight cultures was added to 1 liter of 2XTY medium
containing 100 µg/ml ampicillin and then cultured to
A600 = 0.6 at 37 °C and induced with 2 mM IPTG for 5 h at 37 °C. The cells were pelleted by centrifugation at 5,000 × g for 5 min. and
resuspended in sonic buffer (50 mM sodium phosphate (pH
8.0), 300 mM NaCl) at 5 volumes/gram (wet weight). The
suspension was disrupted by sonic oscillation (30-min bursts/30-min
cooling/30-min bursts at 18 kc) using a Kubota Insonator Model 200 M (Kubota Medical Appliance Supply Co., Tokyo, Japan) at
4 °C. The sonicate was separated by centrifugation at 10,000 × g for 20 min at 4 °C. The precipitate was dissolved in
sonication buffer containing 8 M urea and then applied to a 20-ml column of Ni2+-nitroacetate resin (NTA; Qiagen)
equilibrated with the same buffer. Weakly bound proteins were removed
with buffer A (10 mM Na+, HEPES (pH 7.6), 150 mM NaCl, 10% glycerol, 0.1 mM
phenylmethylsulfonyl fluoride, 10× resin volume). Proteins attached to
the resin were finally eluted with buffer A containing 0.2 M imidazole. Fractions containing proteins were pooled and
sequentially dialyzed against buffer B (20 mM Tris-HCl (pH
7.5), 0.5 M NaCl, 50% glycerol, 2 mM
dithiothreitol, 1 mM EDTA) containing 8, 4, 2, 1, 0.5, and 0.05 M urea. Purified proteins were stored at
30 °C.
/2-shifted squared sine bell function for the t2 dimension
and
/2-shifted sine bell function for the t1
dimension were applied, and zero-filling was applied to acquire real 2K
1K spectra. For two-dimensional 15N-1H
HMQC experiments, the interpulse delay was 4 ms, and 15N
was decoupled according to the GARP (global optimized
alternating-phase rectangular pulses) scheme (24) with a 90 °C pulse
of 150 µs. Free induction decays (16 scans each) of 4 K data
points in the t2 domain were collected for 64 data points in the t1 domain in the
phase-sensitive mode using the States-TPPI method (23). The spectra of
2K × 256 data points were obtained by zero filling the
t1 domain and the
/2-shifted sine bell
function for both the t1 and
t2 dimensions followed by Fourier
transformation. The spectral width for 15N was 100 ppm.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-(1-91) binding upon full size 4.5 S RNA
using a filter binding assay as described by Suzuma et al.
(10). The binding affinity values (M1/2) for Ffh and
EF-G, defined as the concentration that gave half-maximal binding, were
0.15 and 1.5 µM, respectively, indicating that the affinity of EF-G for 4.5 S RNA was lower than that of Ffh by
approximately one order of magnitude.
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Fig. 1.
Predicted secondary structure and nucleotide
sequence of E. coli 4.5 S RNA. The
numbering corresponds to full-length E. coli 4.5 S RNA. Internal bulges are lettered A to E.
A thick line marks the decanucleotide sequence
identical in 4.5 S and 23 S RNAs.
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Fig. 2.
Protein binding activities of wild-type 4.5 S
RNA and site-directed mutants at bulges
A-E. Nucleotide replaced
derivatives of 4.5 S RNA were filter-assayed as described under
"Materials and Methods," and radioactivity retained on filters was
plotted against the concentration of added EF-G.
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Fig. 3.
Protein binding activity of wild-type 4.5 S
RNA and deletion mutants. A, sequences and possible
secondary structures of E. coli 4.5 S RNA and deletion
mutants. Predicted secondary structures of mutants were generated based
on that of wild-type 4.5 S RNA. The numbering corresponds to
full-length E. coli 4.5 S RNA. Internal bulges are lettered
A-E. Boxed sequences of 5 and 10 nucleotides
were added at both the 5' and 3' termini, respectively, to stabilize
the terminal stem structure. B, RNA retention curves from
filter binding assay. Binding activities of wild-type and mutant 4.5 S
RNA were monitored by filter assays as described under "Materials and
Methods." Radioactivity retained on the filter was plotted against
the concentration of EF-G added.
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Fig. 4.
Growth restoration of E. coli
4.5 S RNA conditional mutants S1192 by introducing 4.5 S RNA
derivatives. The growth of S1192-harboring plasmid pKK223-3 (-op)
expressing wild type (WT) ( ) and mutants 1 (
), 2 (
), 3 (
),4 (
), and 5 (
) was monitored by measuring
absorbance at 660 nm.
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Fig. 5.
NMR Spectra of SRP49 and A47U
mutant. Two-dimensional 15N-1H HMQC
spectra of SRP49 (black) and A47U (red) measured
on 500 MHz NMR spectrometer in sodium phosphate buffer (pH 6.3)
containing 5% D2O at 25 °C (a). Imino proton
resonance assignments are denoted by residue numbers. The
new resonances and the missing resonances are denoted by
red and black triangles, respectively. The
secondary structure of SRP49 (b) and proposed secondary
structure of A47U mutant (c). The A47 and U47 residues are
indicated in red.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helix-turn-helix (HTH) motif contained within the five
-helices
of the Ffh M domain. Moreover, this groove was occupied by the
hydrophobic segment of a neighboring M domain. In vitro
chemical protection of 4.5 S RNA by Ffh showed that the protein mainly
protects the 5' side of domain IV, with highly conserved nucleotides
A39, A47, G48, and G49 being the most protected (27). The nucleotides
that are essential for EF-G binding were located opposite those
important for Ffh binding, from the 3' side of bulge A. The EF-G
binding sites within 4.5 S and 23 S rRNA contain the same
decanucleotide sequence. This decamer RNA sequence is included in the
recent reported structure of ribosomal protein L11 in complex with a fragment of 23 S rRNA (32, 33). Comparison of the EF-G binding sites in
4.5 S RNA and 23 S rRNA shows that the last five decanucleotide residues have almost identical conformations (31, 34). In contrast, the
first decanucleotide residues have a distinct orientation, showing
a common structural feature likely to be important for the
recognition of both RNAs by EF-G. To compare the tertiary structure of
the proposed protein binding site, we constructed a model structure of
the A47U mutant in which bulge A forms an A-type helical structure
(Fig. 6), because substitution of A47 with U
caused a loss of binding activity to both Ffh and EF-G. Therefore,
remarkable conformational changes must occur in the binding region of
the A47U mutant. Fig. 6 shows that at least three standard base pairs
are stretched. Moreover, the amino groups of C46 and C62 were involved
in base pairing, and the protruding phosphate backbone that may
interact with proteins as a hydrogen bond acceptor in SRP RNA is
different between the SRP49 and A47U mutant models. Thus, single
changes can cause large conformational changes that may have brought
about the loss of Ffh and EF-G binding affinity in the A47U mutant.
Replacement of A47 with cytosine did not affect the binding activity
(data not shown), suggesting that A47 is important for maintaining
non-canonical helices rather than because it is recognized by
proteins.
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Fig. 6.
Comparison of three-dimensional structural
models of the proposed EF-G binding region. The EF-G binding
region of the A47U mutant (a) and three-dimensional
structure of the corresponding region of SRP49 reproduced from Ref. 29
(b). The strand containing G44-G48 is blue,
C62-C65 is yellow, and A47 and U47 are red. The
model was constructed on an Octane work station (Silicon Graphics) with
the program Insight II (Biosym molecular simulation). U45, which is
believed to bulge outward, is not shown in the model.
Here, we have defined the minimal structure of 4.5 S RNA for binding
EF-G. Taken together with previous data reported by Wood et
al. (17), these data suggest that Ffh and EF-G proteins recognize overlapping domains in the 4.5 S RNA. As mentioned above, an identical decanucleotide sequence is found in the EF-G binding sites of both 4.5 S and 23 S rRNA. Jovine et al. (34) indicated that the last
five decanucleotide residues have almost identical conformations (root
mean square deviation = 0.96 Å). With this structure resemblance, 4.5 S RNA can acquire the ability to compete with 23 S rRNA. Moreover, it is plausible that these sequences in both 4.5 S and 23 S rRNA act as
the first binding site for EF-G. The biological function of the
interaction between 4.5 S RNA and EF-G during translation remains to be
determined. On the basis of all previous data including the structural
comparison, we proposed that 4.5 S RNA might facilitate EF-G-GDP to be
expelled from the ribosome by competing with 23 S rRNA for EF-G binding
(9, 16). However depletion of 4.5 S RNA leads to the rapid inhibition
of translation, and this loss of translation by depletion of 4.5 S RNA
extends to the whole protein (13). Under normal conditions 4.5 S RNA is
not a stable component of the ribosome, but once cells are treated with
fusidic acid or viomycin, 4.5 S RNA comigrates with 70 S ribosome in a sucrose gradient. Under these conditions, distribution of Ffh protein
was unchanged. Moreover, our quantitative analysis demonstrated that
almost all Ffh protein in a cell makes a stable complex, and the
amounts of this complex are unchanged through cell growth (10). Thus,
the function of 4.5 S RNA in translation may be separable from that in
protein secretion. Therefore, even if EF-G and Ffh can compete for 4.5 S RNA binding, this might not contribute to the function of 4.5 S RNA
in translation.
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ACKNOWLEDGEMENTS |
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We thank N. Foster for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science and Sports, and Culture of Japan, by Mitsubishi Chemical Corporation Fund, and in part by the "Research for the Future" program (JSPS-RFTF97L00503) from the Japan Society for the Promotion of Science.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. and Fax:
+81-298-53-6006; E-mail: nakamura.kouji@nifty.ne.jp.
Published, JBC Papers in Press, April 9, 2001, DOI 10.1074/jbc.M101376200
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ABBREVIATIONS |
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The abbreviations used are:
SRP, signal
recognition particle;
bp, base pair(s);
EF-G, elongation factor G;
HMQC, heteronuclear multiquantum coherence;
IPTG, isopropyl--D-thiogalactopyranoside;
NOE, nuclear
Overhauser effect;
NOESY, NOE spectroscopy.
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