A novel GTPase activated by the small subunit of ribosome
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《核酸研究医学期刊》
1 Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Science and 2 Department of Biology, Faculty of Science, Hirosaki University, Hirosaki 036-8561, Japan, 3 The United Graduate School of Agricultural Sciences, Iwate University, Morioka 020-8550, Japan and 4 Department of Biophysics, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
* To whom correspondence should be addressed. Tel: +81 172 39 3592; Fax: +81 172 39 3593; Email: himeno@cc.hirosaki-u.ac.jp
Present addresses: Kyoko Hanawa-Suetsugu, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan
Liliya Kalachnyuk, Biotechnology Research, Institute of Animal Production, S.Z. Gzhytskyi Lviv Academy of Veterinary Medicine, 50 Pekarska Street, Lviv 79010, Ukraine
ABSTRACT
The GTPase activity of Escherichia coli YjeQ, here named RsgA (ribosome small subunit-dependent GTPase A), has been shown to be significantly enhanced by ribosome or its small subunit. The enhancement of GTPase activity was inhibited by several aminoglycosides bound at the A site of the small subunit, but not by a P site-specific antibiotic. RsgA stably bound the small subunit in the presence of GDPNP, but not in the presence of GTP or GDP, to dissociate ribosome into subunits. Disruption of the gene for RsgA from the genome affected the growth of the cells, which predominantly contained the dissociated subunits having only a weak activation activity of RsgA. We also found that 17S RNA, a putative precursor of 16S rRNA, was contained in the small subunit of the ribosome from the RsgA-deletion strain. RsgA is a novel GTPase that might provide a new insight into the function of ribosome.
INTRODUCTION
Translation is accomplished by an elaborate coordination of two subunits of the ribosome, each comprised from one or two RNAs and several dozens of proteins (1). The large subunit has a GTPase-associated center as well as a peptidyl transferase center and three classical tRNA binding sites. Several GTPases are involved in the processes of translation, and their GTP hydrolysis activities are all activated at the GTPase-associated center on the large subunit to introduce a conformational change to themselves and/or ribosome to facilitate translation (2).
Recent progress of genome sequencing has revealed the existence of various GTPases with unknown functions in eubacterial cells (3). yjeQ is an open reading frame encoding a putative GTPase with an unknown function. It can be divided into three domains, the N-terminal domain having a putative OB fold, the central domain having a unique circularly permuted GTPase motif and the C-terminal domain having a unique CxxxxCxHxxxxxC motif that is involved in zinc binding (4). This protein (RsgA) is believed to be essential in Escherichia coli (5), and it has recently been shown to have a weak GTPase activity (6) that is significantly enhanced by the ribosome (7). Unlike other GTPases associated with the ribosome, RsgA was enhanced not by the large subunit but by the small subunit of a ribosome. RsgA is the first factor showing that GTPase activity is activated by the small subunit of a ribosome. In the present study, we show that the activation of RsgA was inhibited by several antibiotics bound around the A site. We further succeeded in disrupting the gene for RsgA from the genome, which significantly affected the growth of the cells and the subunit assembly of ribosomes.
MATERIALS AND METHODS
Preparation of E.coli RsgA
E.coli yjeQ had been cloned into the plasmid pGEMEX2 under the T7 promoter sequence. RsgA was overexpressed in E.coli strain BL21(DE3) by the addition of 1 mM IPTG, and it was purified by a series of column chromatographies using DEAE–cellulose, heparin–Sepharose and Q-Sepharose.
Construction of the yjeQ-disruption strain
The DNA fragment containing yjeQ and its upstream and downstream regions from E.coli W3110 was engineered such that 3' two-thirds of yjeQ are replaced by a gene conferring kanamycin resistance from a plasmid pACYC177. This DNA fragment was transformed into E.coli strain JC7623 (recBC, sbcBC) harboring a plasmid derived from pKO3 (8) carrying yjeQ to replace a part of yjeQ on the genome by the kanamycin-resistant gene. The cells having an expected sequence around yjeQ on the genome were screened from kanamycin-resistant transformants by PCR. The pKO3 derivative carrying yjeQ was removed from the cell by incubation at 42°C, since it has a temperature-sensitive replication origin. The engineered portion of the genome was transferred to E.coli W3110 by phage P1 using kanamycin resistance as a selection marker. The expected recombination of the genome was confirmed both by PCR with several sets of primers and by the direct genome sequencing around yjeQ. The loss of RsgA in the cells was further confirmed by western blotting analysis with anti-RsgA antibody. Finally, we confirmed that a phenotype of slow cell growth of this strain was restored by the introduction of plasmid-encoded RsgA.
Preparation of E.coli ribosome
E.coli A19 cell extract was centrifuged at 20 000 r.p.m. using a Hitachi P-65A rotor for 30 min at 4°C, and the supernatant was centrifuged at 80 000 r.p.m. with Hitachi 80AT rotor for 30 min at 4°C. The pellet was washed with 10 mM Tris–HCl (pH 7.6), 10 mM MgCl2, 1 M NH4Cl and 1 mM DTT, lysed with 10 mM Tris–HCl (pH 7.6), 10 mM MgCl2, 60 mM NH4Cl and 1 mM DTT, and then precipitated through the same buffer solution containing 20% sucrose at 80 000 r.p.m. using a Hitachi 80AT rotor for 30 min at 4°C. The resulting fraction was washed twice with 10 mM Tris–HCl (pH 7.6), 10 mM MgCl2, 1 M NH4Cl and 1 mM DTT, and was stored at –80°C as the ribosome fraction. 70S ribosomes and 50S or 30S subunits were obtained by further centrifugation at 25 000 r.p.m. with a Hitachi P28S rotor for 7 h at 4°C on 5–20% sucrose density gradients containing 20 mM HEPES–KOH (pH 7.5), 6 mM magnesium acetate, 30 mM NH4Cl and 4 mM 2-mercaptoethanol and containing 20 mM HEPES–KOH (pH 7.5), 1 mM magnesium acetate, 200 mM NH4Cl and 4 mM 2-mercaptoethanol, respectively (9).
Assay of GTPase activity
The GTPase reaction proceeded at 37°C in 40 μl reaction mixtures containing 50 mM Tris–HCl (pH 7.5), 200 mM KCl, 5 mM MgCl2, 1 mM DTT, various amounts of RsgA, ribosomes or each subunit and GTP. At each time point, a 4 μl aliquot was withdrawn, and 10 μl of 20 mM EDTA was added to stop the reaction. It was then spotted on a PEI cellulose sheet for thin-layer chromatography. After development using 0.75 M KH2PO4 as a solvent, the radioactivities of the spots of GTP and GDP were monitored using a Bio-Image-Analyzer FLA3000 (Fuji Film).
Sucrose density gradient centrifugation
The reaction mixture or cell extracts were loaded on a 5–20% (w/v) linear sucrose density gradient containing 10 mM Tris–HCl (pH 7.8), 10 mM MgCl2 and 300 mM KCl, and it was centrifuged at 25 000 r.p.m. using a Hitachi P28S rotor for 7 h at 4°C. Proteins in each fraction were precipitated with a 1/3 volume of 100% trichloroacetic acid. After being electrophoresed, the SDS-containing 12% polyacrylamide gel was electroblotted onto a nitrocellulose membrane and then probed with an antibody raised against purified RsgA. The antibody was visualized by chemiluminescence of ECL plus (Amersham) after binding a horseradish peroxidase-conjugated secondary antibody.
RESULTS
The GTPase activity of YjeQ (RsgA) was significantly enhanced by the small subunit of the ribosome
E.coli YjeQ was cloned and overexpressed in E.coli cells. Purified YjeQ had a very weak GTPase activity as already detected (6) (Table 1). When the E.coli ribosome fraction was added to the reaction mixture, the GTPase activity was significantly enhanced. Intriguingly, the GTPase activity was enhanced not only by the 70S ribosome but also by the small (30S) subunit, but it was not enhanced by the large (50S) subunit (Figure 1a). This result is consistent with a recent finding by Daigle and Brown (7). Thus, YjeQ was named here RsgA (ribosome small subunit-dependent GTPase A). The level of GTPase activity of RsgA increased depending on the ribosome concentration, and the addition of a 5-fold molar excess of ribosomes (500 nM) was not sufficient to saturate the GTPase activity (100 nM) (Figure 1b).
Table 1. Kinetic parameters for GTPase activity of E.coli RsgA
Figure 1. Activation of GTPase activity by the ribosome. (a) GTPase activity of RsgA was activated by 70S ribosome (open rectangles) or the 30S subunit (closed circles) but not by the 50S subunit (open circles): 100 nM RsgA, 54 nM ribosome or its subunits and 50 μM GTP were used. The ribosome or its subunits used had no detectable GTPase activity in the absence of RsgA. (b) The GTPase activity of RsgA was dependent on the concentration of ribosome: 100 nM RsgA, various concentrations of 70S ribosome and 50 μM GTP were used.
We measured the kinetic parameters in the presence and absence of 54 nM 70S ribosome or the small subunit. Both of them increased the GTPase activity of RsgA in terms of kcat/Km by two orders of magnitude (Table 1). The enhancement of GTPase activity was largely due to the increased kcat. Enhancement of GTPase activity was not observed when 16S or 23S rRNA was used instead of the ribosome or when the ribosome was treated with RNase A, indicating that enhancement of GTPase activity requires both RNA and protein components of the ribosome.
The ribosome-dependent GTPase activity was affected by the A site-specific antibiotics
The effects of several antibiotics on the enhancement of GTPase activity of RsgA by the ribosome were examined (Figure 2). As expected, chloramphenicol, which binds to the peptidyl-transferase center of the large subunit, did not inhibit the enhancement of GTPase activity. In contrast, aminoglycoside antibiotics such as neomycin, paromomycin, kanamycin and gentamycin, which bind specifically to the helix 44 at the A site of the decoding region of the small subunit to induce miscoding (10), significantly affected the ribosome-dependent GTPase activity. The addition of 5–500 μM of these aminoglycosides resulted in a 50% decrease in the level of the ribosome-dependent GTPase activity. Inhibition of the ribosome-dependent GTPase activity was also observed when a higher concentration (5 mM) of streptomycin or tetracyclin, which also binds around the A site (11,12), was added. These findings suggest that RsgA is in close contact with the A site of the decoding region of the small subunit. On the other hand, neither kasugamycin, a P site-specific antibiotic (13), nor spectinomycin, which binds helix 34 in the head of the small subunit (10), had effect on the ribosome-dependent GTPase activity of RsgA.
Figure 2. Inhibition of ribosome-dependent GTPase activity of RsgA by antibiotics. Various concentrations of neomycin (open diamonds), paromomycin (open rectangles), gentamycin (open triangles), kanamycin (closed circles), tetracyclin (hatched rectangles), streptomycin (open circles), kasugamycin (hatched triangles), spectinomycin (closed triangle) and chloramphenicol (closed rectangles) were added to the GTPase reaction mixtures each containing 300 nM RsgA, 54 nM ribosomes and 50 μM GTP. Ribosome-independent GTPase activity of RsgA was not inhibited by 5 mM of each antibiotic.
RsgA stably bound the small subunit in the presence of GDPNP, but not in the presence of GTP or GDP, to dissociate ribosome into subunits
To examine the association between RsgA and the ribosome in vitro, a mixture of RsgA and ribosomes was fractionated by sucrose density gradient centrifugation, and the complex formation was evaluated using an antibody raised against RsgA. Almost no or only a small population of RsgA co-sedimented with 70S ribosome or its subunits in the absence of any guanine nucleotides. This was also the case in the presence of GTP or GDP (Figure 3a and b). The ratio of the levels of 70S, 50S and 30S fractions was not changed regardless of the presence or absence of GTP or GDP. In contrast, RsgA predominantly co-sedimented with the small subunit in the presence of GDPNP, a nonhydrolyzable GTP analogue (Figure 3c). In addition, almost all of the ribosomes were dissociated into subunits in the presence of GDPNP. These results strongly suggest that the GTP form of RsgA binds the 30S subunit, probably around the subunit interface, and induces a conformational change in the small subunit or the 70S ribosome to transiently loosen the association between the two subunits. Subsequent GTP hydrolysis may result in a conformational change in RsgA so that it immediately dissociates from the ribosome to restore the stable association between the two subunits of the ribosome. Daigle and Brown (7) have shown that RsgA binds the 30S subunit in the presence of not only GDPNP but also GDP, and that the interaction in the presence of GDP depends on the salt concentration. This is consistent with the result of the present study that was performed at 300 mM KCl.
Figure 3. Association between RsgA and the ribosome. Two μM ribosomes was incubated with 5 μM RsgA at 37°C in a 100 μl solution containing 80 mM Tris–HCl (pH 7.8), 150 mM ammonium chloride, 7 mM magnesium acetate, 2.5 mM DTT and (a) 1 mM GTP, (b) 1 mM GDP, (c) 1 mM GDPNP or (d) 1 mM GDPNP + 500 μM neomycin. After 10 min of incubation, the reaction mixture was fractionated by sucrose density gradient centrifugation, and RsgA in each fraction was detected with an antibody raised against RsgA after electrophoresis.
We also examined the effect of neomycin on the association between RsgA and the ribosome. When 500 μM neomycin, which is sufficient to inhibit the enhancement of GTPase activity of the ribosome (Figure 2), was added to the reaction mixture, neither dissociation into subunits nor binding of RsgA to the small subunit was observed in the presence of GDPNP (Figure 3d). This indicates that aminoglycosides inhibit the step of binding of the GTP form of RsgA to the ribosome rather than GTPase activation after binding to the ribosome or the dissociation of the GDP form of RsgA from the ribosome.
The majority of 70S ribosomes were dissociated into subunits in RsgA-deficient cells
To determine the role of RsgA in cells, we tried to disrupt the gene for RsgA from the genome. Since E.coli yjeQ has been reported to be an essential gene (5), we initially attempted to make a cell in which yjeQ has been put on a plasmid derived from pKO3 (8) and yjeQ on the genome has been replaced by a kanamycin-resistant gene. This plasmid, having a temperature-sensitive replication origin, could be removed from the cell by incubation at 42°C if yjeQ is not essential for the cell. Unexpectedly, the cells were viable at 42°C. We have no idea about the reason for a failure in disruption of yjeQ in a previous study (5). The complete loss of RsgA in the cells was confirmed both by PCR with several sets of primers and by western blotting analysis with anti-RsgA antibody.
The doubling time of the RsgA-deletion strain was 2.34 ± 0.05-fold longer than that of the wild type at incubation temperatures of 42, 37 and 30°C (Table 2). The ratio did not change within the range of these temperatures. The prolonged doubling time was restored by the introduction of plasmid-encoded RsgA.
Table 2. Effect of deletion of yjeQ on cell growth
We then compared the pattern of ribosomes from RsgA-deficient cells with that from wild-type cells by sucrose density gradient centrifugation. Intriguingly, the majority of 70S ribosomes were dissociated into subunits in RsgA-deficient cells (Figure 4a and b). The introduction of RsgA into RsgA-deficient cells using a plasmid restored the normal pattern of ribosomes (Figure 4c). Similar patterns were observed when cells were grown at 30 or 42°C. These findings indicate that the deficiency of RsgA resulted in a reduction in the level of translation activity in the cell, probably leading to the phenotype of slow cell growth.
Figure 4. Effect of RsgA on subunit formation of ribosomes. Extracts from W3110 cells (a), RsgA-deficient cells (b) and W3110 cells harboring a low-copy plasmid pMW119 carrying yjeQ (c) grown at 37°C were fractionated by sucrose density gradient centrifugation.
The 30S fraction from RsgA-deficient cells had only a weak GTPase activity of RsgA
We prepared a 30S fraction from RsgA-deficient cells under the condition of a low magnesium concentration (1 mM magnesium acetate) in which 70S ribosomes were completely dissociated into subunits and found that it enhanced the GTPase activity of RsgA less efficiently than that from wild-type cells (Figure 5a). Then, we examined a 30S fraction prepared under the condition of a high magnesium concentration (6 mM magnesium acetate) and the 30S fraction prepared from 70S ribosomes under low magnesium concentration. Both 30S fractions prepared via 70S ribosomes from wild-type and RsgA-deficient cells were highly active in the enhancement of the GTPase activity of RsgA (Figure 5a). The 30S fraction prepared from wild-type cells under the condition of a high magnesium concentration had slightly less ability to enhance GTPase activity of RsgA (Figure 5a, left). The 30S fraction prepared from RsgA-deficient cells under the condition of a high magnesium concentration had much less ability (Figure 5a, right). These results indicate that some population of small subunits that do not participate in 70S ribosome particles have no capacity to enhance the GTPase activity of RsgA even in wild-type cells, and that deprivation of RsgA significantly increases the size of such a population. It is possible that such a population is an immature form of small subunits.
Figure 5. Activation abilities of GTPase activity of RsgA of small subunits from various preparations and rRNAs that they contain. (a) Activation of GTPase activities by 30S fractions from W3110 cells (left) or RsgA-deficient cells (right) prepared under the condition of a high magnesium concentration (6 mM magnesium acetate) (open circles), under the condition of a low magnesium concentration (1 mM magnesium acetate) (hatched circles) and under the condition of a low magnesium concentration via 70S ribosome (closed circles): 100 nM RsgA, 54 nM ribosome or its subunits and 50 μM GTP were used. Each preparation of ribosome had no detectable GTPase activity in the absence of RsgA. (b) Electrophoresis of rRNAs from various preparations of cell extracts and small subunits on a 6% polyacrylamide gel containing 7 M urea. Cell extracts (lanes 1–3) and 70S (lanes 4 and 7) or 30S fractions (lanes 5, 6, 8 and 9) were from wild-type cells of mid-log phase (lanes 1, 2, 5 and 6), RsgA-deficient cells of mid-log phase (lanes 3, 8 and 9). 30S fractions were prepared under the condition of a low magnesium concentration via 70S ribosomes (lanes 5 and 8) and under the condition of a high magnesium concentration (lanes 6 and 9). Cell extracts were prepared from wild-type cells with 2 h culture after the addition of 34 μg/ml chloramphenicol when OD600 reached 0.5 (lane 2). An unidentified band below the band of 16S rRNA is indicated by an asterisk.
To identify the difference in the small subunit between wild-type and RsgA-deficient cells, we prepared RNA components from the 30S fractions prepared under various conditions, and compared them by polyacrylamide gel electrophoresis (Figure 5b). Both 30S fractions via 70S ribosomes from wild-type and RsgA-deficient cells, as well as that prepared from wild-type cells under high magnesium condition, almost exclusively contain apparently normal size of 16S rRNA. On the other hand, the 30S fraction prepared under high magnesium condition from RsgA-deficient cells contained a band of slower mobility than 16S rRNA in addition to the band of apparently normal 16S rRNA (Figure 5b, lane 9). It has been shown that 17S rRNA, a precursor of 16S rRNA, is accumulated when the protein synthesis is inhibited by chloramphenicol (14). We confirmed that the mobility of this extra band is almost the same as that of 17S rRNA prepared from wild-type cells cultured with chloramphenicol (Figure 5b). An unidentified band below the band of 16S rRNA was observed in the 30S fraction prepared from RsgA-deficient cells under the condition of a high magnesium concentration (Figure 5b, lane 9). It may be a degradation product from 16S or 17S rRNA.
DISCUSSION
Various GTPases are involved in the processes of eubacterial translation, and their GTP hydrolysis activities are all activated at the GTPase-associated center on the large subunit. In the present study, we demonstrated that the GTPase activity of RsgA is significantly enhanced by the small subunit of the ribosome. Several other GTPases with unidentified functions have been shown to interact with ribosomes (3), although very few have been shown to interact with the small subunit (15). To the best of our knowledge, RsgA is the first molecule showing that GTPase activity is activated by the small subunit of a ribosome. It was also suggested that RsgA transiently binds around the subunit interface on the small subunit just during GTP hydrolysis (Figure 3). RsgA can be characterized by three highly conserved motifs, the N-terminal OB fold, the central GTPase motif that is circularly permuted and the C-terminal CxxxxCxHxxxxxC motif that is involved in zinc binding (4). The molecular mechanism underlying the involvement of such a unique structure in GTPase activation by the small subunit is yet to be clarified.
The ribosome-dependent GTPase activity of RsgA as well as its binding to the small subunit was found to be inhibited by several A site-specific antibiotics, suggesting that RsgA has a role around the A site of the decoding region. It is thus possible that RsgA plays a role in the processes of translation. The finding of predominant dissociation of ribosomes by deprivation of RsgA (Figure 4) reinforces the notion that RsgA is involved not only in translation of some special mRNA.
GTP hydrolysis has the potential to facilitate conformational change in the molecule and/or its contact molecule to modulate their interaction. The present results suggest that binding the GTP form of RsgA around the A site of the subunit interface on the small subunit induces a conformational change in the small subunit or the 70S ribosome to transiently loosen the association between the two subunits, and subsequent GTP hydrolysis results in a conformational change in RsgA so that it immediately dissociates from the ribosome to restore the stable association between the two subunits of the ribosome. Apparently, the ribosome preferentially binds the GTP form of RsgA, indicating that it acts as a GTPase-activating protein. During the translation processes, the small subunit of ribosome undergoes conformational change to ensure the fidelity of decoding (16). Conformational change in the ribosome might also be required in other situations, such as in the mRNA landing on the small subunit to start the initiation step of translation or in the unwinding of the mRNA secondary structure to properly position mRNA in a single-stranded form at the decoding region. Messenger RNA must pass through the narrow downstream tunnel to accomplish the initiation and elongation processes even if it is highly structured, and thereby the requirement of an as-yet-unidentified mechanism has been postulated to overcome such difficulties (17).
A recent study has shown that the truncation of the N-terminal OB fold of RsgA affects the interaction with the ribosome (7). RsgA has a unique zinc-binding motif that is potentially involved in the second RNA binding. We have found that RsgA has the capacity to bind RNA with broad specificity, although the binding evenly occurs regardless of the presence or absence of GTP or GDP (unpublished results). Like the activation of RsgA, the initiation process of translation occurs not only on the small subunit but also on 70S ribosome (18). In the classical initiation process, the A site of the small subunit is believed to be occupied by IF-1 in which the OB fold is the site of binding (19,20). The functional relationship between RsgA and classical initiation factors, especially IF-1, has yet to be determined.
Some proteins having a putative GTP binding motif have been shown to be involved in ribosome biogenesis in eukaryotes (21–24). Era, a eubacterial GTPase that can bind the E.coli 30S subunit or 16S rRNA, is also likely to be involved in ribosome maturation (15). In the present study, a depletion of RsgA caused accumulation of a fraction of immature small subunits in the cells, raising the possibility that RsgA functions in a step of maturation of the ribosome. If this is the case, the precursor of the 30S subunit accumulated in RsgA-deficient cells should be a substrate for RsgA. However, RsgA preferred apparently mature small subunits from 70S ribosomes to the small subunits that do not participate in the 70S ribosome particles in the RsgA-deficient cells (Figure 5). Furthermore, not only the small subunit but also 70S ribosome was a good substrate for RsgA (Figure 1, Table 1). Note that the accumulation of immature rRNA (17S rRNA) can be a result of a defect in the de novo synthesis of ribosomal proteins by the addition of chloramphenicol (14). Further studies will be required to conclude whether RsgA is involved in maturation of the small subunit or translation processes.
RsgA is widely distributed among the prokaryotic kingdom. Its homologue is also present in Arabidopsis but not in yeast or animals. It could be categorized into a new type of GTPase that would provide a new insight into the function of ribosome.
ACKNOWLEDGEMENTS
We thank Dr G.M. Church for providing pKO3. We also thank Gene Research Center of Hirosaki University for the use of the facility. This work was supported by grant-in-aids for scientific research from the Ministry of Education, Science, Sports and Culture, Japan to A.M. (No. 14035201) and H.H. (No. 14035202, 15013201 and 15032202), grant-in-aids for scientific research from the Japan Society for the Promotion of Science to A.M. (No. 14380322) and H.H. (No. 14360044), a Human Frontier Science Program research grant (RG0291/2000-M 100) to H.H., a research grant from Hirosaki University to A.M. and H.H. and Matsumae International Foundation Research Fellowship Program to L.K.
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* To whom correspondence should be addressed. Tel: +81 172 39 3592; Fax: +81 172 39 3593; Email: himeno@cc.hirosaki-u.ac.jp
Present addresses: Kyoko Hanawa-Suetsugu, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan
Liliya Kalachnyuk, Biotechnology Research, Institute of Animal Production, S.Z. Gzhytskyi Lviv Academy of Veterinary Medicine, 50 Pekarska Street, Lviv 79010, Ukraine
ABSTRACT
The GTPase activity of Escherichia coli YjeQ, here named RsgA (ribosome small subunit-dependent GTPase A), has been shown to be significantly enhanced by ribosome or its small subunit. The enhancement of GTPase activity was inhibited by several aminoglycosides bound at the A site of the small subunit, but not by a P site-specific antibiotic. RsgA stably bound the small subunit in the presence of GDPNP, but not in the presence of GTP or GDP, to dissociate ribosome into subunits. Disruption of the gene for RsgA from the genome affected the growth of the cells, which predominantly contained the dissociated subunits having only a weak activation activity of RsgA. We also found that 17S RNA, a putative precursor of 16S rRNA, was contained in the small subunit of the ribosome from the RsgA-deletion strain. RsgA is a novel GTPase that might provide a new insight into the function of ribosome.
INTRODUCTION
Translation is accomplished by an elaborate coordination of two subunits of the ribosome, each comprised from one or two RNAs and several dozens of proteins (1). The large subunit has a GTPase-associated center as well as a peptidyl transferase center and three classical tRNA binding sites. Several GTPases are involved in the processes of translation, and their GTP hydrolysis activities are all activated at the GTPase-associated center on the large subunit to introduce a conformational change to themselves and/or ribosome to facilitate translation (2).
Recent progress of genome sequencing has revealed the existence of various GTPases with unknown functions in eubacterial cells (3). yjeQ is an open reading frame encoding a putative GTPase with an unknown function. It can be divided into three domains, the N-terminal domain having a putative OB fold, the central domain having a unique circularly permuted GTPase motif and the C-terminal domain having a unique CxxxxCxHxxxxxC motif that is involved in zinc binding (4). This protein (RsgA) is believed to be essential in Escherichia coli (5), and it has recently been shown to have a weak GTPase activity (6) that is significantly enhanced by the ribosome (7). Unlike other GTPases associated with the ribosome, RsgA was enhanced not by the large subunit but by the small subunit of a ribosome. RsgA is the first factor showing that GTPase activity is activated by the small subunit of a ribosome. In the present study, we show that the activation of RsgA was inhibited by several antibiotics bound around the A site. We further succeeded in disrupting the gene for RsgA from the genome, which significantly affected the growth of the cells and the subunit assembly of ribosomes.
MATERIALS AND METHODS
Preparation of E.coli RsgA
E.coli yjeQ had been cloned into the plasmid pGEMEX2 under the T7 promoter sequence. RsgA was overexpressed in E.coli strain BL21(DE3) by the addition of 1 mM IPTG, and it was purified by a series of column chromatographies using DEAE–cellulose, heparin–Sepharose and Q-Sepharose.
Construction of the yjeQ-disruption strain
The DNA fragment containing yjeQ and its upstream and downstream regions from E.coli W3110 was engineered such that 3' two-thirds of yjeQ are replaced by a gene conferring kanamycin resistance from a plasmid pACYC177. This DNA fragment was transformed into E.coli strain JC7623 (recBC, sbcBC) harboring a plasmid derived from pKO3 (8) carrying yjeQ to replace a part of yjeQ on the genome by the kanamycin-resistant gene. The cells having an expected sequence around yjeQ on the genome were screened from kanamycin-resistant transformants by PCR. The pKO3 derivative carrying yjeQ was removed from the cell by incubation at 42°C, since it has a temperature-sensitive replication origin. The engineered portion of the genome was transferred to E.coli W3110 by phage P1 using kanamycin resistance as a selection marker. The expected recombination of the genome was confirmed both by PCR with several sets of primers and by the direct genome sequencing around yjeQ. The loss of RsgA in the cells was further confirmed by western blotting analysis with anti-RsgA antibody. Finally, we confirmed that a phenotype of slow cell growth of this strain was restored by the introduction of plasmid-encoded RsgA.
Preparation of E.coli ribosome
E.coli A19 cell extract was centrifuged at 20 000 r.p.m. using a Hitachi P-65A rotor for 30 min at 4°C, and the supernatant was centrifuged at 80 000 r.p.m. with Hitachi 80AT rotor for 30 min at 4°C. The pellet was washed with 10 mM Tris–HCl (pH 7.6), 10 mM MgCl2, 1 M NH4Cl and 1 mM DTT, lysed with 10 mM Tris–HCl (pH 7.6), 10 mM MgCl2, 60 mM NH4Cl and 1 mM DTT, and then precipitated through the same buffer solution containing 20% sucrose at 80 000 r.p.m. using a Hitachi 80AT rotor for 30 min at 4°C. The resulting fraction was washed twice with 10 mM Tris–HCl (pH 7.6), 10 mM MgCl2, 1 M NH4Cl and 1 mM DTT, and was stored at –80°C as the ribosome fraction. 70S ribosomes and 50S or 30S subunits were obtained by further centrifugation at 25 000 r.p.m. with a Hitachi P28S rotor for 7 h at 4°C on 5–20% sucrose density gradients containing 20 mM HEPES–KOH (pH 7.5), 6 mM magnesium acetate, 30 mM NH4Cl and 4 mM 2-mercaptoethanol and containing 20 mM HEPES–KOH (pH 7.5), 1 mM magnesium acetate, 200 mM NH4Cl and 4 mM 2-mercaptoethanol, respectively (9).
Assay of GTPase activity
The GTPase reaction proceeded at 37°C in 40 μl reaction mixtures containing 50 mM Tris–HCl (pH 7.5), 200 mM KCl, 5 mM MgCl2, 1 mM DTT, various amounts of RsgA, ribosomes or each subunit and GTP. At each time point, a 4 μl aliquot was withdrawn, and 10 μl of 20 mM EDTA was added to stop the reaction. It was then spotted on a PEI cellulose sheet for thin-layer chromatography. After development using 0.75 M KH2PO4 as a solvent, the radioactivities of the spots of GTP and GDP were monitored using a Bio-Image-Analyzer FLA3000 (Fuji Film).
Sucrose density gradient centrifugation
The reaction mixture or cell extracts were loaded on a 5–20% (w/v) linear sucrose density gradient containing 10 mM Tris–HCl (pH 7.8), 10 mM MgCl2 and 300 mM KCl, and it was centrifuged at 25 000 r.p.m. using a Hitachi P28S rotor for 7 h at 4°C. Proteins in each fraction were precipitated with a 1/3 volume of 100% trichloroacetic acid. After being electrophoresed, the SDS-containing 12% polyacrylamide gel was electroblotted onto a nitrocellulose membrane and then probed with an antibody raised against purified RsgA. The antibody was visualized by chemiluminescence of ECL plus (Amersham) after binding a horseradish peroxidase-conjugated secondary antibody.
RESULTS
The GTPase activity of YjeQ (RsgA) was significantly enhanced by the small subunit of the ribosome
E.coli YjeQ was cloned and overexpressed in E.coli cells. Purified YjeQ had a very weak GTPase activity as already detected (6) (Table 1). When the E.coli ribosome fraction was added to the reaction mixture, the GTPase activity was significantly enhanced. Intriguingly, the GTPase activity was enhanced not only by the 70S ribosome but also by the small (30S) subunit, but it was not enhanced by the large (50S) subunit (Figure 1a). This result is consistent with a recent finding by Daigle and Brown (7). Thus, YjeQ was named here RsgA (ribosome small subunit-dependent GTPase A). The level of GTPase activity of RsgA increased depending on the ribosome concentration, and the addition of a 5-fold molar excess of ribosomes (500 nM) was not sufficient to saturate the GTPase activity (100 nM) (Figure 1b).
Table 1. Kinetic parameters for GTPase activity of E.coli RsgA
Figure 1. Activation of GTPase activity by the ribosome. (a) GTPase activity of RsgA was activated by 70S ribosome (open rectangles) or the 30S subunit (closed circles) but not by the 50S subunit (open circles): 100 nM RsgA, 54 nM ribosome or its subunits and 50 μM GTP were used. The ribosome or its subunits used had no detectable GTPase activity in the absence of RsgA. (b) The GTPase activity of RsgA was dependent on the concentration of ribosome: 100 nM RsgA, various concentrations of 70S ribosome and 50 μM GTP were used.
We measured the kinetic parameters in the presence and absence of 54 nM 70S ribosome or the small subunit. Both of them increased the GTPase activity of RsgA in terms of kcat/Km by two orders of magnitude (Table 1). The enhancement of GTPase activity was largely due to the increased kcat. Enhancement of GTPase activity was not observed when 16S or 23S rRNA was used instead of the ribosome or when the ribosome was treated with RNase A, indicating that enhancement of GTPase activity requires both RNA and protein components of the ribosome.
The ribosome-dependent GTPase activity was affected by the A site-specific antibiotics
The effects of several antibiotics on the enhancement of GTPase activity of RsgA by the ribosome were examined (Figure 2). As expected, chloramphenicol, which binds to the peptidyl-transferase center of the large subunit, did not inhibit the enhancement of GTPase activity. In contrast, aminoglycoside antibiotics such as neomycin, paromomycin, kanamycin and gentamycin, which bind specifically to the helix 44 at the A site of the decoding region of the small subunit to induce miscoding (10), significantly affected the ribosome-dependent GTPase activity. The addition of 5–500 μM of these aminoglycosides resulted in a 50% decrease in the level of the ribosome-dependent GTPase activity. Inhibition of the ribosome-dependent GTPase activity was also observed when a higher concentration (5 mM) of streptomycin or tetracyclin, which also binds around the A site (11,12), was added. These findings suggest that RsgA is in close contact with the A site of the decoding region of the small subunit. On the other hand, neither kasugamycin, a P site-specific antibiotic (13), nor spectinomycin, which binds helix 34 in the head of the small subunit (10), had effect on the ribosome-dependent GTPase activity of RsgA.
Figure 2. Inhibition of ribosome-dependent GTPase activity of RsgA by antibiotics. Various concentrations of neomycin (open diamonds), paromomycin (open rectangles), gentamycin (open triangles), kanamycin (closed circles), tetracyclin (hatched rectangles), streptomycin (open circles), kasugamycin (hatched triangles), spectinomycin (closed triangle) and chloramphenicol (closed rectangles) were added to the GTPase reaction mixtures each containing 300 nM RsgA, 54 nM ribosomes and 50 μM GTP. Ribosome-independent GTPase activity of RsgA was not inhibited by 5 mM of each antibiotic.
RsgA stably bound the small subunit in the presence of GDPNP, but not in the presence of GTP or GDP, to dissociate ribosome into subunits
To examine the association between RsgA and the ribosome in vitro, a mixture of RsgA and ribosomes was fractionated by sucrose density gradient centrifugation, and the complex formation was evaluated using an antibody raised against RsgA. Almost no or only a small population of RsgA co-sedimented with 70S ribosome or its subunits in the absence of any guanine nucleotides. This was also the case in the presence of GTP or GDP (Figure 3a and b). The ratio of the levels of 70S, 50S and 30S fractions was not changed regardless of the presence or absence of GTP or GDP. In contrast, RsgA predominantly co-sedimented with the small subunit in the presence of GDPNP, a nonhydrolyzable GTP analogue (Figure 3c). In addition, almost all of the ribosomes were dissociated into subunits in the presence of GDPNP. These results strongly suggest that the GTP form of RsgA binds the 30S subunit, probably around the subunit interface, and induces a conformational change in the small subunit or the 70S ribosome to transiently loosen the association between the two subunits. Subsequent GTP hydrolysis may result in a conformational change in RsgA so that it immediately dissociates from the ribosome to restore the stable association between the two subunits of the ribosome. Daigle and Brown (7) have shown that RsgA binds the 30S subunit in the presence of not only GDPNP but also GDP, and that the interaction in the presence of GDP depends on the salt concentration. This is consistent with the result of the present study that was performed at 300 mM KCl.
Figure 3. Association between RsgA and the ribosome. Two μM ribosomes was incubated with 5 μM RsgA at 37°C in a 100 μl solution containing 80 mM Tris–HCl (pH 7.8), 150 mM ammonium chloride, 7 mM magnesium acetate, 2.5 mM DTT and (a) 1 mM GTP, (b) 1 mM GDP, (c) 1 mM GDPNP or (d) 1 mM GDPNP + 500 μM neomycin. After 10 min of incubation, the reaction mixture was fractionated by sucrose density gradient centrifugation, and RsgA in each fraction was detected with an antibody raised against RsgA after electrophoresis.
We also examined the effect of neomycin on the association between RsgA and the ribosome. When 500 μM neomycin, which is sufficient to inhibit the enhancement of GTPase activity of the ribosome (Figure 2), was added to the reaction mixture, neither dissociation into subunits nor binding of RsgA to the small subunit was observed in the presence of GDPNP (Figure 3d). This indicates that aminoglycosides inhibit the step of binding of the GTP form of RsgA to the ribosome rather than GTPase activation after binding to the ribosome or the dissociation of the GDP form of RsgA from the ribosome.
The majority of 70S ribosomes were dissociated into subunits in RsgA-deficient cells
To determine the role of RsgA in cells, we tried to disrupt the gene for RsgA from the genome. Since E.coli yjeQ has been reported to be an essential gene (5), we initially attempted to make a cell in which yjeQ has been put on a plasmid derived from pKO3 (8) and yjeQ on the genome has been replaced by a kanamycin-resistant gene. This plasmid, having a temperature-sensitive replication origin, could be removed from the cell by incubation at 42°C if yjeQ is not essential for the cell. Unexpectedly, the cells were viable at 42°C. We have no idea about the reason for a failure in disruption of yjeQ in a previous study (5). The complete loss of RsgA in the cells was confirmed both by PCR with several sets of primers and by western blotting analysis with anti-RsgA antibody.
The doubling time of the RsgA-deletion strain was 2.34 ± 0.05-fold longer than that of the wild type at incubation temperatures of 42, 37 and 30°C (Table 2). The ratio did not change within the range of these temperatures. The prolonged doubling time was restored by the introduction of plasmid-encoded RsgA.
Table 2. Effect of deletion of yjeQ on cell growth
We then compared the pattern of ribosomes from RsgA-deficient cells with that from wild-type cells by sucrose density gradient centrifugation. Intriguingly, the majority of 70S ribosomes were dissociated into subunits in RsgA-deficient cells (Figure 4a and b). The introduction of RsgA into RsgA-deficient cells using a plasmid restored the normal pattern of ribosomes (Figure 4c). Similar patterns were observed when cells were grown at 30 or 42°C. These findings indicate that the deficiency of RsgA resulted in a reduction in the level of translation activity in the cell, probably leading to the phenotype of slow cell growth.
Figure 4. Effect of RsgA on subunit formation of ribosomes. Extracts from W3110 cells (a), RsgA-deficient cells (b) and W3110 cells harboring a low-copy plasmid pMW119 carrying yjeQ (c) grown at 37°C were fractionated by sucrose density gradient centrifugation.
The 30S fraction from RsgA-deficient cells had only a weak GTPase activity of RsgA
We prepared a 30S fraction from RsgA-deficient cells under the condition of a low magnesium concentration (1 mM magnesium acetate) in which 70S ribosomes were completely dissociated into subunits and found that it enhanced the GTPase activity of RsgA less efficiently than that from wild-type cells (Figure 5a). Then, we examined a 30S fraction prepared under the condition of a high magnesium concentration (6 mM magnesium acetate) and the 30S fraction prepared from 70S ribosomes under low magnesium concentration. Both 30S fractions prepared via 70S ribosomes from wild-type and RsgA-deficient cells were highly active in the enhancement of the GTPase activity of RsgA (Figure 5a). The 30S fraction prepared from wild-type cells under the condition of a high magnesium concentration had slightly less ability to enhance GTPase activity of RsgA (Figure 5a, left). The 30S fraction prepared from RsgA-deficient cells under the condition of a high magnesium concentration had much less ability (Figure 5a, right). These results indicate that some population of small subunits that do not participate in 70S ribosome particles have no capacity to enhance the GTPase activity of RsgA even in wild-type cells, and that deprivation of RsgA significantly increases the size of such a population. It is possible that such a population is an immature form of small subunits.
Figure 5. Activation abilities of GTPase activity of RsgA of small subunits from various preparations and rRNAs that they contain. (a) Activation of GTPase activities by 30S fractions from W3110 cells (left) or RsgA-deficient cells (right) prepared under the condition of a high magnesium concentration (6 mM magnesium acetate) (open circles), under the condition of a low magnesium concentration (1 mM magnesium acetate) (hatched circles) and under the condition of a low magnesium concentration via 70S ribosome (closed circles): 100 nM RsgA, 54 nM ribosome or its subunits and 50 μM GTP were used. Each preparation of ribosome had no detectable GTPase activity in the absence of RsgA. (b) Electrophoresis of rRNAs from various preparations of cell extracts and small subunits on a 6% polyacrylamide gel containing 7 M urea. Cell extracts (lanes 1–3) and 70S (lanes 4 and 7) or 30S fractions (lanes 5, 6, 8 and 9) were from wild-type cells of mid-log phase (lanes 1, 2, 5 and 6), RsgA-deficient cells of mid-log phase (lanes 3, 8 and 9). 30S fractions were prepared under the condition of a low magnesium concentration via 70S ribosomes (lanes 5 and 8) and under the condition of a high magnesium concentration (lanes 6 and 9). Cell extracts were prepared from wild-type cells with 2 h culture after the addition of 34 μg/ml chloramphenicol when OD600 reached 0.5 (lane 2). An unidentified band below the band of 16S rRNA is indicated by an asterisk.
To identify the difference in the small subunit between wild-type and RsgA-deficient cells, we prepared RNA components from the 30S fractions prepared under various conditions, and compared them by polyacrylamide gel electrophoresis (Figure 5b). Both 30S fractions via 70S ribosomes from wild-type and RsgA-deficient cells, as well as that prepared from wild-type cells under high magnesium condition, almost exclusively contain apparently normal size of 16S rRNA. On the other hand, the 30S fraction prepared under high magnesium condition from RsgA-deficient cells contained a band of slower mobility than 16S rRNA in addition to the band of apparently normal 16S rRNA (Figure 5b, lane 9). It has been shown that 17S rRNA, a precursor of 16S rRNA, is accumulated when the protein synthesis is inhibited by chloramphenicol (14). We confirmed that the mobility of this extra band is almost the same as that of 17S rRNA prepared from wild-type cells cultured with chloramphenicol (Figure 5b). An unidentified band below the band of 16S rRNA was observed in the 30S fraction prepared from RsgA-deficient cells under the condition of a high magnesium concentration (Figure 5b, lane 9). It may be a degradation product from 16S or 17S rRNA.
DISCUSSION
Various GTPases are involved in the processes of eubacterial translation, and their GTP hydrolysis activities are all activated at the GTPase-associated center on the large subunit. In the present study, we demonstrated that the GTPase activity of RsgA is significantly enhanced by the small subunit of the ribosome. Several other GTPases with unidentified functions have been shown to interact with ribosomes (3), although very few have been shown to interact with the small subunit (15). To the best of our knowledge, RsgA is the first molecule showing that GTPase activity is activated by the small subunit of a ribosome. It was also suggested that RsgA transiently binds around the subunit interface on the small subunit just during GTP hydrolysis (Figure 3). RsgA can be characterized by three highly conserved motifs, the N-terminal OB fold, the central GTPase motif that is circularly permuted and the C-terminal CxxxxCxHxxxxxC motif that is involved in zinc binding (4). The molecular mechanism underlying the involvement of such a unique structure in GTPase activation by the small subunit is yet to be clarified.
The ribosome-dependent GTPase activity of RsgA as well as its binding to the small subunit was found to be inhibited by several A site-specific antibiotics, suggesting that RsgA has a role around the A site of the decoding region. It is thus possible that RsgA plays a role in the processes of translation. The finding of predominant dissociation of ribosomes by deprivation of RsgA (Figure 4) reinforces the notion that RsgA is involved not only in translation of some special mRNA.
GTP hydrolysis has the potential to facilitate conformational change in the molecule and/or its contact molecule to modulate their interaction. The present results suggest that binding the GTP form of RsgA around the A site of the subunit interface on the small subunit induces a conformational change in the small subunit or the 70S ribosome to transiently loosen the association between the two subunits, and subsequent GTP hydrolysis results in a conformational change in RsgA so that it immediately dissociates from the ribosome to restore the stable association between the two subunits of the ribosome. Apparently, the ribosome preferentially binds the GTP form of RsgA, indicating that it acts as a GTPase-activating protein. During the translation processes, the small subunit of ribosome undergoes conformational change to ensure the fidelity of decoding (16). Conformational change in the ribosome might also be required in other situations, such as in the mRNA landing on the small subunit to start the initiation step of translation or in the unwinding of the mRNA secondary structure to properly position mRNA in a single-stranded form at the decoding region. Messenger RNA must pass through the narrow downstream tunnel to accomplish the initiation and elongation processes even if it is highly structured, and thereby the requirement of an as-yet-unidentified mechanism has been postulated to overcome such difficulties (17).
A recent study has shown that the truncation of the N-terminal OB fold of RsgA affects the interaction with the ribosome (7). RsgA has a unique zinc-binding motif that is potentially involved in the second RNA binding. We have found that RsgA has the capacity to bind RNA with broad specificity, although the binding evenly occurs regardless of the presence or absence of GTP or GDP (unpublished results). Like the activation of RsgA, the initiation process of translation occurs not only on the small subunit but also on 70S ribosome (18). In the classical initiation process, the A site of the small subunit is believed to be occupied by IF-1 in which the OB fold is the site of binding (19,20). The functional relationship between RsgA and classical initiation factors, especially IF-1, has yet to be determined.
Some proteins having a putative GTP binding motif have been shown to be involved in ribosome biogenesis in eukaryotes (21–24). Era, a eubacterial GTPase that can bind the E.coli 30S subunit or 16S rRNA, is also likely to be involved in ribosome maturation (15). In the present study, a depletion of RsgA caused accumulation of a fraction of immature small subunits in the cells, raising the possibility that RsgA functions in a step of maturation of the ribosome. If this is the case, the precursor of the 30S subunit accumulated in RsgA-deficient cells should be a substrate for RsgA. However, RsgA preferred apparently mature small subunits from 70S ribosomes to the small subunits that do not participate in the 70S ribosome particles in the RsgA-deficient cells (Figure 5). Furthermore, not only the small subunit but also 70S ribosome was a good substrate for RsgA (Figure 1, Table 1). Note that the accumulation of immature rRNA (17S rRNA) can be a result of a defect in the de novo synthesis of ribosomal proteins by the addition of chloramphenicol (14). Further studies will be required to conclude whether RsgA is involved in maturation of the small subunit or translation processes.
RsgA is widely distributed among the prokaryotic kingdom. Its homologue is also present in Arabidopsis but not in yeast or animals. It could be categorized into a new type of GTPase that would provide a new insight into the function of ribosome.
ACKNOWLEDGEMENTS
We thank Dr G.M. Church for providing pKO3. We also thank Gene Research Center of Hirosaki University for the use of the facility. This work was supported by grant-in-aids for scientific research from the Ministry of Education, Science, Sports and Culture, Japan to A.M. (No. 14035201) and H.H. (No. 14035202, 15013201 and 15032202), grant-in-aids for scientific research from the Japan Society for the Promotion of Science to A.M. (No. 14380322) and H.H. (No. 14360044), a Human Frontier Science Program research grant (RG0291/2000-M 100) to H.H., a research grant from Hirosaki University to A.M. and H.H. and Matsumae International Foundation Research Fellowship Program to L.K.
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