A physiological connection between tmRNA and peptidyl-tRNA hydrolase f
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《核酸研究医学期刊》
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560 012, India
* To whom correspondence should be addressed. Tel: +91 80 2293 2686; Fax: +91 80 2360 2697; Email: varshney@mcbl.iisc.ernet.in
ABSTRACT
The bacterial ssrA gene codes for a dual function RNA, tmRNA, which possesses tRNA-like and mRNA-like regions. The tmRNA appends an oligopeptide tag to the polypeptide on the P-site tRNA by a trans-translation process that rescues ribosomes stalled on the mRNAs and targets the aberrant protein for degradation. In cells, processing of the stalled ribosomes is also pioneered by drop-off of peptidyl-tRNAs. The ester bond linking the peptide to tRNA is hydrolyzed by peptidyl-tRNA hydrolase (Pth), an essential enzyme, which releases the tRNA and the aberrant peptide. As the trans-translation mechanism utilizes the peptidyl-transferase activity of the stalled ribosomes to free the tRNA (as opposed to peptidyl-tRNA drop-off), the need for Pth to recycle such tRNAs is bypassed. Thus, we hypothesized that tmRNA may rescue a defect in Pth. Here, we show that overexpression of tmRNA rescues the temperature-sensitive phenotype of Escherichia coli (pthts). Conversely, a null mutation in ssrA enhances the temperature-sensitive phenotype of the pthts strain. Consistent with our hypothesis, overexpression of tmRNA results in decreased accumulation of peptidyl-tRNA in E.coli. Furthermore, overproduction of tmRNA in E.coli strains deficient in ribosome recycling factor and/or lacking the release factor 3 enhances the rescue of pthts strains. We discuss the physiological relevance of these observations to highlight a major role of tmRNA in decreasing cellular peptidyl-tRNA load.
INTRODUCTION
For various physiological reasons, not all ribosomes that begin to translate an mRNA reach the termination codon. A significant population of the translating ribosomes stalls in between the decoding cycles (1,2). Stalled ribosomes pose a serious impediment to mRNA translation as they deplete pools of free tRNA and ribosomes, bringing protein synthesis to a halt. One of the cellular strategies to process stalled ribosomes involves peptidyl-tRNA drop-off (Figure 1, upper) (3–5). While the mechanisms responsible for the drop-off of peptidyl-tRNAs from the ribosomes are not very well understood (6), genetic and biochemical evidence (7–10) suggests that in E.coli, ribosome recycling factor (RRF) and EFG, as well as other factors such as RF3, IF1, IF2 and RelA contribute to this phenomenon. The peptidyl-tRNAs are recycled by peptidyl-tRNA hydrolase (Pth), which hydrolyzes the ester link between the tRNA and the peptide (11). As accumulation of peptidyl-tRNAs leads to cellular toxicity, Pth is an essential protein in bacteria (12,13).
Figure 1. Pathways depicting release of the tRNAs from the peptidyl-tRNAs by processing of stalled ribosomes pioneered either by dissociation of peptidyl-tRNAs (upper) or by tmRNA-mediated trans-translation (lower). Stalled ribosomes for trans-translation may arise either by endonucleolytic cleavage of mRNA in stalled ribosomes or from the translation of the truncated mRNAs.
Another cellular strategy that leads to the rescue of ribosomes stalled on fragmented mRNAs, involves trans-translation (14). Here the product of ssrA gene, tmRNA (also known as SsrA), is aminoacylated by alanyl-tRNA synthetase, and recruited to the ribosomal A site with the help of EFTu and an ancillary protein, SmpB. The peptidyl-transferase activity of the ribosome transfers the peptide from the P-site bound peptidyl-tRNA to the alanine on the –CCA end of the tmRNA. Consequently, the tRNA that was sequestered as peptidyl-tRNA in the stalled complex is released (15). The elongation process then resumes to translate a 30 nt long open reading frame (mRNA portion) of the tmRNA to encode the ANDENYALAA sequence tag (in E.coli). When the ribosomes reach the termination codon in the tmRNA, the C-terminally tagged peptide is released by the activity of release factors, and subjected to degradation by cellular proteases (16–18). Interestingly, as the tmRNA-mediated mechanism utilizes the peptidyl-transferase activity of the stalled ribosomes to liberate tRNA (as opposed to peptidyl-tRNA drop-off), the need for Pth to recycle such tRNAs is bypassed (Figure 1, lower). Since, a number of cellular processes result in accumulation of stalled ribosomes with fragmented mRNA (19–23), trans-translation may represent a major mechanism to recycle both the stalled ribosomes and the tRNAs. This raises an important question as to whether excess tmRNA in E.coli can alleviate the requirement for Pth activity.
Earlier studies have shown that RRF and RF3 are involved in peptidyl-tRNA drop-off from the ribosome. Thus, conditional lethal strains of E.coli, with a temperature-sensitive phenotype because of a mutation in pth could be rescued by downregulation of RRF expression (8). Conversely, overproduction of RRF in E.coli is toxic under conditions of limiting Pth activity (7). It was also observed that disruption of the prfC gene coding for RF3 resulted in suppression of the temperature-sensitive phenotype of a pthts strain. In vitro analyses suggested that disruption of prfC and downregulation of RRF result in decreased levels of peptidyl-tRNA drop-off (8). Also, the tRNALys appears to be one of the first species of tRNAs that is depleted by its sequestration as peptidyl-tRNA in the pthts strains of E.coli. Overproduction of this tRNA increases the temperature threshold at which the strains continue to grow (4,24).
Here, we show that overproduction of tmRNA rescues the temperature-sensitive phenotype of a pthts strain and that a major cellular function of tmRNA is to lessen the peptidyl-tRNA load. Further, coupling of tmRNA overproduction with downregulation of RRF and depletion of RF3, results in a stronger suppression of pthts. However, the role of Pth in E.coli is central and it was not possible to dispense with pth function even upon integration of the known suppressors in a single strain.
MATERIALS AND METHODS
Plasmids, strains and growth conditions
Plasmids and strains used in this study are listed in Table 1. Luria–Bertani (LB) liquid or solid (with 1.5% agar) media (Difco, USA) were used for growth (25). The medium was supplemented with various antibiotics at the following final concentrations: tetracycline, 7.5 μg/ml; kanamycin, 25 μg/ml; chloramphenicol, 30 μg/ml; and ampicillin, 100 μg/ml as required.
Table 1. List of plasmids/strains
Cloning
All constructs were made using standard recombinant DNA techniques (25) as follows:
E.coli ssrA gene
DNA primers 5'-GAAAAGCTTATTGGCTATCAC-3' and 5'-CACTAAGCTTGATTAACAGAG-3' containing HindIII sites were used to amplify the ssrA gene (along with its promoter) from E.coli HB101 DNA using Pfu DNA polymerase by PCR consisting of 30 cycles of incubations at 95°C for 1 min, 40°C for 30 s and 72°C for 2 min, digested with HindIII and cloned between the same sites of pTrc99C.
E.coli smpB-ssrA
The linked smpB and ssrA genes (along with their promoters) were PCR amplified from E.coli HB101 DNA by Pfu DNA polymerase using 5'-AACGGATCCCGGCGCTGGGT-3', and 5'-CACTAAGCTTGATTAACAGAG-3' primers containing BamHI and HindIII sites, respectively, by 30 cycles of incubations at 95°C for 1 min, 45°C for 30 s and 72°C for 2 min, digested with BamHI and HindIII, and cloned into similarly digested pTrc99C.
E.coli valU operon and generation of pTrc-valU-ssrA construct
The valU operon containing tRNALys gene was cloned from E.coli HB101 DNA by PCR using Pfu DNA polymerase with 5'-GCGAATTCTTTAGAAAATGC-3' and 5'-CAACTGGATCCTGCTGCAGGTTGG-3' primers, involving 30 cycles of incubations at 95°C for 1 min, 50°C for 30 s and 72°C for 1 min 20 s. The PCR product was digested with EcoRI and BamHI, and cloned between the same sites of pTrc-ssrA to obtain pTrc-valU-ssrA construct. The pTrc-valU construct was generated by deleting ssrA gene from the pTrc-valU-ssrA by digestion with HindIII followed by religation with T4 DNA ligase. The veracity of the clone was established by its ability to complement E.coli AA7852 for its pthts phenotype.
E.coli and Bacillus subtilis pth genes
The E.coli pth gene along with its promoter was amplified from E.coli HB101 using 5'-CACGTCTTGAAGGCAAACGG-3' and 5'-CGAACAACGTGACCGATCG-3' primers, digested with EcoRI and EcoRV, and cloned into the same sites of pTrc99C. Similarly, the open reading frame of B.subtilis pth gene was PCR amplified from B.subtilis genomic DNA using 5'-CGCCATGGTTGATTGCCGGTC-3' and 5'-CATTGAAGCTTCGAGACTGCC-3' primers. The PCR reaction involved 30 cycles of heating at 94°C for 1 min, 50°C for 45 s and 70°C for 1 min 45 s. The PCR product was digested with NcoI and HindIII, and cloned between the same sites of pTrc99C and pACDH.
Generation of E.coli strains
All strains described below were generated following the method of Datsenko and Wanner (30).
E.coli AA7852ssrA::Kn and E.coli CP78ssrA::Kn strains
pTrc-smpB-ssrA was digested with EcoRV and religated to delete a small fragment containing unwanted sequences of the plasmid and smpB origin to generate pTrc-smpB-ssrA*. This was digested with PvuII to cleave within the ssrA gene for cloning of a HincII DNA fragment carrying KnR cassette from pUC4K. This construct (pTrc-ssrA::Kn) was digested with EcoRV–HincII to obtain a 2.1 kb DNA containing the disrupted ssrA gene for electroporation into E.coli DY330. The ssrA disruption in E.coli DY330 was confirmed by PCR using flanking primers. The ssrA::Kn locus from the DY330 strain was mobilized into E.coli AA7852 (pthts) and E.coli CP78 (pth+, parent strain of AA7852) by P1-mediated transductions (31).
E.coli AA7852 prfC::Kn, E.coli CP79 prfC::Kn and E.coli CP79 prfC strains
The KnR cassette from pKD4 was amplified with 5'-CGCGGCGCGATGCCGCTTACTCAAGAAAGAATTGTGTAGGCTGGAGCTGCTTCG-3' and 5'-CAGGGCGCAGCGCGTACCGCGCCCTGGAGGCAAGAACATATGAATATCCTCCTTA-3' primers using Pfu DNA polymerase and electroporated into E.coli TG1 harboring pKD46. Transformants were screened for replacement of prfC with KnR cassette by PCR with the flanking primers (5'-GTCGAGCCAGGTAAACTC-3' and 5'-GCGACGGCAGAGGTCAAC-3') followed by digestion with PstI. The prfC::Kn was subsequently mobilized into E.coli AA7852 and E.coli CP79 by P1-mediated transductions to generate E.coli AA7852 prfC::Kn and E.coli CP79 prfC::Kn strains. The E.coli AA7852 prfC and E.coli CP79 prfC strains were obtained by excising the KnR cassette in the presence of pCP20 expressing the FLP recombinase (30,32).
E.coli AA7852 frr1 and E.coli CP79 frr1 strains
The strain (frr1) wherein RRF expression was down regulated was generated by insertion of a CmR cassette at the –32 position of frr promoter. The CmR cassette was PCR amplified from pKD3 using 5'-GGCAAATAAAATAGCCTAATAATCCAGACGATTACCCGGTGTAGGCTGGAGCTGCTTC-3' and 5'-GGAAGTGTGCTAAGTATAGCCCTGATTAAACATATTACATATGAATATCCTCCTTA-3' primers and electroporated into E.coli DY330. Integration of the CmR cassette was verified by PCR using flanking primers (5'-CGCAGCAACCGG-AGCGTAC-3' and 5'-GTTTCGCACTCGCTTCATCG-3'). E.coli AA7852 frr1 and E.coli CP79 frr1 strains were generated by P1-mediated transductions.
E.coli CP79 prfC, frr1
This strain was generated by P1-mediated transduction of frr1 locus (CmR) from AA7852frr1 to CP79prfC.
Generation of E.coli pth::kan
The KnR cassette was amplified from pUC4K using 5'-CTCCGCTGCGCGAAGAGGCTAAATTCTTTGGTTATACAAAGCCACGTTGTGTCTC-3' and 5'-CAGATCCAGTTCGTCGTGGGCCACCAGAATTTCGTCCGCGCTGAGGTCTGCCTCGTG-3' primers and electroporated into E.coli DY330 harboring pACDBsuPth. Transformants (KnR) were checked for disruption of the pth gene by PCR using flanking primers (5'-GTACGGATCCACGATTAAATTGATTGTCGGCCTG-3' and 5'-AGACAAGCTTTTATTGCGCTTTAAAGGCGTGCAA-3').
Analysis of SsrA expression
Total RNA was prepared from E.coli (33), electrophoresed on a 1.5% agarose gel and vacuum blotted to nytran membrane (25). The blot was probed with 5' 32P-labeled DNA oligomer (5'-GTTTCGCAAATCCCGTCGAAT-3') complementary to SsrA. The same blot was then probed for a second time with 5' 32P-labeled DNA oligomer (5'-TACCATCGGCGCTACGGCGTTTC-3') complementary to 5S rRNA (25). Blots were subjected to BioImage analysis (FLA2000, Fuji).
Analysis of peptidyl-tRNAs
Luria–Bertani medium (5 ml) containing ampicillin was inoculated with 2% inoculum from overnight cultures and grown at 30°C to an OD of 0.6 (595 nm), and shifted to 37°C for 45 min. The cells were then chilled on wet ice. All the subsequent steps for the isolation of total tRNA under acidic conditions at 4°C, separation on acid urea gels and electroblotting onto nytran membrane were as described in (34). The blot was probed (7) with a 5' 32P-labeled DNA complementary to tRNAHis, and tRNATyr (5'-GACAACTGGAATCACAATCC-3' and 5'-TACAGTCTGCTCCCTTTGGCCGCTC-3', respectively) and subjected to BioImage analysis (FLA2000, Fuji) using storage phosphor imager plates (BAS SR, Fuji).
Immunoblotting
The cell-free extracts from log-phase cultures were separated on 15% SDS–PAGE, transferred to PVDF membrane and probed with anti-RRF antibodies (7).
Analysis of essentiality of pth
Phage P1 lysate was raised on E.coli DY330 (pth::Kn) harboring pACDBsuPth and used to transduce E.coli CP79prfC, CP79frr1, and CP79prfC, frr1 harboring pTrc99C, pTrcBsuPth, pTrc-ssrA or pTrc-valU-ssrA. The transductants (KnR) were screened by PCR for pth locus with the primers (5'-CACGTCTTGAAGGCAAACGG-3' and 5'-CGAACAACGTGACCGATCG-3').
RESULTS
Overproduction of tmRNA (SsrA) rescues the temperature-sensitive phenotype of E.coli AA7852 (pthts)
To test the hypothesis if overproduction of tmRNA could rescue the pthts phenotype of E.coli AA7852, we introduced into this strain, various plasmid constructs (Table 1) by genetic transformation and checked for the growth of the transformants at different temperatures. As shown in Figure 2A, true to its genotype, the strain AA7852 harboring the pTrc99C vector alone grew at the permissive and semi-nonpermissive temperatures of 30 and 37°C, respectively but ceased to grow as the temperature was raised to 40°C (sectors 2). As a control, when the strain harbored a plasmid-borne copy of E.coli pth gene (pTrcEcoPth), it grew at 40°C (sectors 1). Interestingly, a plasmid-borne copy of ssrA gene (sectors 3) also rescued the growth of AA7852 at 40°C (compare sectors 2 and 3). Simultaneous presence of the extrachromosomal copy of smpB gene (coding for SmpB protein) along with ssrA (pTrc-ssrA-smpB) conferred no apparent advantage towards rescue by plasmid-borne ssrA (compare sectors 3 and 4), and was not analyzed any further. To examine the extent of tmRNA overproduction in the transformants, we analyzed the levels of tmRNA and 5S rRNA . Based on the relative levels of tmRNA and 5S rRNA , in the transformants harboring plasmid-borne copies of ssrA (pTrc-ssrA or pTrc-ssrA-smpB), production of tmRNA was about 4.5-fold higher than the chromosomal background suggesting that a moderate level of overproduction of tmRNA lessens the severity of the pthts phenotype in E.coli.
Figure 2. (A) Analysis of complementation of E.coli AA7852 (pthts) with ssrA. Various plasmids were introduced into E.coli AA7852 and the transformants were streaked on LB agar plates containing ampicillin, at various temperatures indicated above the plates. Sectors: 1, pTrcEcoPth; 2, pTrc99C (vector); 3, pTrc-ssrA; and 4, pTrc-ssrA-smpB. (B) Expression of tmRNA (SsrA) in E.coli AA7852 harboring different plasmids indicated above the lanes (or a ssrA::Kn null strain) was detected by northern blotting using radiolabeled DNA probes for tmRNA and 5 S rRNA . Relative levels of tmRNA (tmRNA/5S rRNA) shown in panel iii were obtained by quantification of signals by BioImage analyzer. Relative levels of tmRNA were normalized to transformants harboring pTrc99C vector (which was set as 1).
E.coli AA7852 (pthts) with ssrA::Kn allele are hypersensitive to temperature
In another approach to demonstrate that the chromosomally encoded tmRNA contributes to rescue of the pthts phenotype, we disrupted the ssrA gene in the E.coli AA7852 with Kn marker to generate E.coli AA7852ssrA::Kn strain. This procedure resulted in the complete absence of tmRNA in the AA7852ssrA::Kn strain . As seen in Figure 3A (sectors 3), the AA7852ssrA::Kn strain became hypersensitive to temperature and was not viable at 37°C, a temperature at which the parent strain AA7852 sustained good growth (Figure 2A, sector 2). Introduction of the plasmid-borne copy of ssrA into the AA7852ssrA::Kn strain, rescued its growth not only at 37°C but also at 40°C (Figure 3A, sectors 4) ruling out that lack of growth of AA7852ssrA::Kn strain at the semi-nonpermissive temperature was due to some non-specific effects emanating from the alterations at the chromosomal level. Further, as shown in Figure 3B, in liquid cultures also, while at 30°C, the AA7852ssrA::Kn strain harboring the vector alone, a plasmid-borne copy of ssrA or pth genes grew with similar rates; at 39°C, the AA7852ssrA::Kn strain harboring vector alone failed to show any significant growth. However, the growth of the strain at 39°C was rescued by the presence of a plasmid-borne copy of ssrA or pth. Expectedly, the rescue by pth was better than that by ssrA. In a control experiment, when the ssrA locus was disrupted in E.coli CP78 (pth+ isogenic strain of AA7852) in exactly the same manner, under the same conditions, no adverse effects of ssrA disruption were noticed (Figure 3A, compare sectors 1 and 2). Taken together, the observations in Figures 2 and 3 clearly suggest that ssrA is a suppressor of pthts phenotype in E.coli.
Figure 3. (A) Analysis of growth of E.coli CP78 (pth+, ssrA::Kn) harboring pTrc99C or pTrc-ssrA (sectors 1 and 2, respectively) and E.coli AA7852 (pthts, ssrA::Kn) harboring pTrc99C or pTrc-ssrA (sectors 3 and 4, respectively) on LB plates containing ampicillin at various temperatures indicated above the plates. (B) Analysis of growth in LB medium. Overnight cultures (0.05%) were inoculated into fresh medium and grown under shaking conditions at 30 or 39°C. Progress in growth was monitored by taking absorbance of samples at 595 nm at different times.
Overexpression of tmRNA lowers the steady-state levels of peptidyl-tRNAs in E.coli AA7852
To understand the mechanism of tmRNA-mediated rescue of the pthts phenotype, we analyzed the effect of its overexpression on the levels of peptidyl-tRNAs (peptidyl-tRNAHis and peptidyl-tRNATyr) in the cells. To ensure preservation of the sensitive ester bond that links amino acid or the peptide to the tRNA, RNA was prepared under cold and acidic conditions, and analyzed using acid urea gels. In these gels, tRNAs attached to peptides of heterogeneous length migrate slower than the aminoacylated tRNA, often in the form of a smear (7). Further, since the absolute levels of total tRNA vary in the preparations, to compare changes in the steady-state levels of peptidyl-tRNAs, their abundance as percent fraction of total tRNA (for tRNAHis or tRNATyr) was determined within each sample. As shown in Figure 4A, expectedly, at the permissive temperature of 30°C where the Pth is active, none of the transformants accumulate any significant levels of peptidyl-tRNAHis (lanes 1, 3 and 5). However, at the semi-nonpermissive temperature of 37°C, while the transformant harboring vector alone accumulated 54% of the tRNAHis as peptidyl-tRNAHis (lane 4), the presence of ssrA construct in the strain decreased this to 37% (lane 6). Expressing a wild-type copy of pth in AA7852, reduced the extent of accumulation of peptidyl-tRNAHis at 37°C to 11% (lane 2). Similar results were obtained with tRNATyr (Figure 4B). At 37°C, accumulation of tRNATyr as peptidyl-tRNATyr was 32% in transformants harboring vector alone (lane 4) which was decreased to 20% in transformants harboring the ssrA gene (lane 6). The level of peptidyl-tRNATyr in the transformants with plasmid-borne copy of wild-type pth at 37°C, was 9% (lane 2). Thus, consistent with our hypothesis of ssrA serving as a suppressor of pthts, overproduction of tmRNA at 37°C resulted in decreased accumulation of both the tRNATyr and tRNAHis in the form of peptidyl-tRNAs.
Figure 4. Analysis of the accumulation of peptidyl-tRNAs in E.coli AA7852 (pthts) in the presence of vector (pTrc99C) or pTrc-ssrA. Transformants were grown at permissive (30°C) or semi-nonpermissive (37°C) temperatures. Total tRNA was prepared under acidic conditions, fractionated on acid-urea gel, transferred to nytran membrane and hybridized with 5' end 32P-labeled anti-tRNAHis (A) or anti-tRNATyr (B) probes. Signals were quantified by BioImage analyzer (Fuji) and % peptidyl-tRNA was calculated as . These are shown below the lanes.
ssrA-mediated suppression of pthts in prfC, and frr promoter down backgrounds
Heurgue-Hamard et al. (8) reported that disruption of prfC (RF3), or the promoter region of frr (RRF) resulted in suppression of pthts phenotype in E.coli. In order to integrate these results and the tmRNA effect described above, and to further our understanding on the mechanisms that lead to suppression of the pthts phenotype, we generated a prfC derivative of AA7852 by replacement of the open reading frame of RF3 with KnR marker, and a promoter down frr by insertion of CmR marker at –32 position in the promoter region (frr1). Genetic manipulations in both strains were confirmed by PCR analysis (Materials and Methods). Further, an immunoblot analysis of the cell-free extracts obtained from the frr1 derivative showed downregulation of expression of RRF . Yet another derivative of AA7852 that we generated possessed both the prfC and frr1 genetic alterations. As expected, when compared with the parent strain AA7852 (Figure 6, sector 2), all the three derivatives of AA7852 (prfC, frr1 and the double mutation of prfC and frr1) rescued the phenotype of the pthts strain (sectors 4, 6 and 8, respectively, at 40 and 42°C). More importantly, introduction of a plasmid-borne ssrA gene in all of these strains resulted in a higher efficiency of suppression of pthts in AA7852 (compare sectors 4, 6 and 8 with 3, 5 and 7, respectively at 42°C). At 42°C, it is seen that rescue by prfC and frr1 markers is somewhat better than that conferred by overproduction of tmRNA (compare sectors 1 with 4 and 6, respectively). Nevertheless, integration of prfC, frr1 with a plasmid-borne copy of ssrA (sector 7) resulted in a remarkable rescue of E.coli AA7852.
Figure 5. Immunoblot analysis of total cell-free extracts of E.coli AA7852 (lane 2) and its frr1 derivative (lane 3) for RRF. Cell-free extracts (10 μg) were analyzed by SDS–PAGE and detected by Coomassie blue stain . A similar gel was prepared for transfer to PVDF membrane and immunoblotted using anti-RRF antibodies . Lane 1 contains pure RRF as marker.
Figure 6. Analysis of growth of E.coli AA7852 (sectors 1 and 2), and its derivatives E.coli AA7852prfC::Kn (sectors 3 and 4), E.coli AA7852frr1 (sectors 5 and 6), and E.coli AA7852frr1, prfC::Kn (sectors 7 and 8) harboring pTrc99C (sectors 2, 4, 6 and 8) or pTrc-ssrA (sectors 1, 3, 5 and 7) at various temperatures as indicated on LB agar plates containing ampicillin.
pth is essential in E.coli
With the identification of tmRNA as an additional suppressor of pth, there are now at least five factors (RRF, RF3, RelA, tRNALys and tmRNA) whose influence on Pth is well characterized in E.coli. Of these, RF3, RRF and RelA are responsible for increased drop-off of peptidyl-tRNA, and tmRNA leads to a decrease in the level of peptidyl-tRNA. On the other hand, tRNALys (in valU operon) functions at a step downstream of the peptidyl-tRNA release. These observations, together with the fact that both of the identifiable pth genes in Saccharomyces cerevisiae could be deleted, raise the question of whether the pth gene in E.coli can be dispensed with. To address this, we first integrated one or many of the conditions that lead to rescue of pthts, into E.coli CP79 (relA–). We also generated an E.coli strain wherein 0.1 kb segment of the chromosomal pth open reading frame was replaced with a kanamycin resistance marker (pth::Kn) in the presence of a helper plasmid harboring a pth gene of B.subtilis origin under the control of an E.coli promoter (Figure 7A). Phage P1 lysate was raised on this strain to attempt knockout of the pth gene in the E.coli CP79 derivatives by transduction. As shown in Table 2, a number of 1000 transductants on a Kn plate with a control strain harboring plasmid-borne pth gene of B.subtilis origin suggests a high efficiency of tranduction. However, despite many attempts, the number of transductants on the test strains, which lacked a pth support plasmid but into which various suppressors of pth had been integrated, was very low (Table 2). Further, PCR screening (Figure 7A) of the entire test transductants obtained, revealed that they either possessed an intact copy, or both an intact and a disrupted copy of the pth genes in their chromosomes (a representative gel is shown in Figure 7B, and data are summarized in Table 2). On the other hand, the control transductants (obtained in the presence of support plasmid) possessed only a disrupted copy of the E.coli pth gene (Figure 7B, lane 1). Use of several other strains of E.coli in such experiments yielded identical results (data not shown). Taken together, these observations suggest that pth plays a central role and is indispensable in E.coli.
Figure 7. Analysis of pth gene disruption in E.coli. (A) Schematic representation of the pth locus and its disruption by Kn cassette in E.coli. Expected sizes of the PCR products from chromosomal DNAs of an intact and disrupted loci are as indicated. (B) PCR screening of representative transductants. Lanes: M, DNA size markers ( DNA digested with HindII and HindIII); WT, wild-type locus; lane 1, positive control transductant (disrupted locus); lanes 2–7, transductants obtained on CP79prfC, frr1, pTrc-ssrA (lanes 2 and 3), CP79 frr1, pTrc-ssrA (lanes 4 and 5) and CP79prfC, frr1, pTrc-valU-ssrA (lanes 6 and 7) backgrounds.
Table 2. Analysis of pth gene disruption in E.coli
DISCUSSION
In bacteria, the product of ssrA gene, SsrA is a well-conserved molecule of 360 nt in length. In E.coli, SsrA was originally identified as 10Sa RNA upon further fractionation of the 10S RNA (35). Subsequently, SsrA was shown to function both as tRNA and mRNA, hence the new name tmRNA (14). The 3' end of tmRNA terminates with a –CCA sequence to which alanine is ligated by alanyl-tRNA synthetase. The alanylated-tmRNA is recruited to the ribosomal A-sites of the stalled ribosomes for the process of trans-translation, which attaches an AANDENYALAA sequence (in E.coli) to the C-terminal of the incomplete peptides and directs the aberrant proteins for degradation by the cellular proteases (4,36–38). In this study, we have uncovered yet another facet of tmRNA function in decreasing the peptidyl-tRNA load in the cells.
Ribosomes may stall on either a full-length or a truncated mRNA. While the stalled complexes with truncated mRNAs (with no or very short 3' extensions) are direct and preferred substrates for tmRNA, even those with an intact mRNA may be directed to trans-translation following the action of codon-specific ribonuclease (19–22). The observation that in the wild-type ssrA background, overproduction of tmRNA raises the threshold of the temperature sensitivity of the pthts strain suggests that in the cells, there exists a population of stalled ribosomes, which is the substrate for trans-translation, but remains unprocessed by the available tmRNA. Consequently, this population of stalled ribosomes (which may possess either a truncated or intact mRNA) must be processed via alternate pathways resulting in dissociation of peptidyl-tRNAs from them, which in turn should be processed by Pth to recycle the tRNAs. Thus, we believe that rescue of the pthts strain AA7852, by overproduction of tmRNA is due to diversion of this population of the stalled ribosomes from dropping-off peptidyl-tRNAs to engaging them in trans-translation (Figure 1, lower). This will in turn, decrease the substrate load on Pth, and make the available Pth activity sufficient to process peptidyl-tRNAs that arise from other physiological processes. Further, although the exact mechanisms by which RRF and RF3 result in peptidyl-tRNA drop-off are not understood, it is clear that downregulation of RRF and/or absence of RF3 result in decreased peptidyl-tRNA load in the cell (7,8). Interestingly, as shown in Figure 6, overproduction of tmRNA in a strain harboring frr1 and prfC mutations in single or in combination, results in a synergistic effect on the rescue of the pthts phenotype. The synergism between the two mutations themselves and with ssrA suggests that the stalled ribosomes are channeled to utilization by all the three factors and, while the RRF and RF3 pathways rescue them by drop-off of peptidyl-tRNAs (Figure 1, upper), ssrA rescues them by trans-translation (Figure 1, lower).
Studies on mutants of E.coli and its phages have unraveled crucial aspects of the host's physiology (39). It was observed that rap (same as pth) mutation in E.coli resulted in defective growth of bacteriophage (40,41). Interestingly, yet another independent study, reported a similar phenotype for the sip (same as ssrA) mutation in E.coli (42,43). While no link has been established between these two separate studies, these observations support the importance of ssrA and the physiological relevance of the connection between ssrA and pth that we have established in this study. In fact, at least in Neisseria gonorrhoeae, the ssrA is an essential gene (44). It will be interesting to investigate the status of peptidyl-tRNA/Pth in this organism.
The genetic and biochemical analyses of the suppressor mutations (bar mutations) in phage that rescue the growth on rap (pth) mutants of E.coli have shown that the phage-encoded minigenes (wild-type bar regions, at least one of which is located in the imm region) are responsible for excess drop-off of the peptidyl-tRNAs and depleting the availability of tRNA for translation (45). Such peptidyl-tRNAs are recycled by Pth. Hence, the effect of Pth on phage growth is a direct one. Whether the effect of ssrA on phage growth is also a direct one (e.g. by trans-translation) is not clear. At least, the lack of tmRNA-mediated targeting of aberrant proteins to degradation is not responsible for the bacteriophage growth defect in sip (ssrA) mutants (38). On the other hand, based on recent reports that the ribosomes with truncated mRNAs with no or very short 3' extensions are the real substrates for trans-translation by tmRNA (46,47) and that the minigene toxicity is more a consequence of frequent re-initiation events (48), it appears that the ssrA effect on bacteriophage growth is an indirect one. The presence of ssrA most likely decreases peptidyl-tRNA load by engaging the stalled ribosomes into trans-translation (thereby withdrawing the chances of the factor-dependent or spontaneous processes that result in drop-off of peptidyl-tRNAs). This process, frees Pth to now hydrolyze the peptidyl-tRNAs arising out of the minigenes of the bacteriophage , or other host activities. However, this interpretation does not rule out a direct role for ssrA in the bacteriophage growth. Recently, it has been shown that at least within certain codon contexts, much of the peptidyl-tRNA population arising from minigene expression remained in the ribosomes (49). Such ‘stand-by’ stalled ribosomal complexes can be further processed to become substrates for trans-translation (19–22). Therefore, it will be interesting to study the mechanism by which ssrA contributes to the lambdoid phage growth.
Finally, biochemical studies show that Saccharomyces cerevisiae possesses at least two distinct types of Pth activities similar to those found in bacteria and archaea. Recently, it was shown that deletions in S.cerevisiae of the two identifiable pth genes (individually or in combination) were viable (50,51). A large number of factors in E.coli (RRF, RF3, IF1, IF2, EFG, tmRNA, RelA, tRNALys, etc.) affect peptidyl-tRNA levels in cell. And, the fact that many of these have also been characterized as suppressors of Pth suggests that the role of Pth in E.coli (and possibly in other organisms) is central to the process of mRNA translation. On the other hand, the knowledge of a large number of suppressors that decrease the peptidyl-tRNA load in E.coli also makes it desirable to attempt to delete the only known pth gene in E.coli. Thus, we integrated many of the known suppressor markers in E.coli to attempt deletion of pth gene. However, our studies (Figure 7) clearly support the fact that the Pth activity is indeed an essential function. In fact, as has also been suggested by the authors of the S.cerevisiae study (51), there may well be several Pth-like activities in eukaryotes to recycle tRNAs. In rabbit reticulocyte lysates, one of the activities that recycles tRNAs from the peptidyl-tRNAs is a phosphodiesterase that cleaves between the C and A, the last two nucleotides in the tRNA (52). The tRNA is subsequently repaired by tRNA nucleotidyltransferase (53). Whether such Pth activity is present in yeast is not known. Certainly, the reaffirmation of the essentiality of Pth activity in E.coli is a good indicator of the presence of additional Pth activities in yeast.
ACKNOWLEDGEMENTS
We thank Dr U. L. RajBhandary and our laboratory colleagues for their suggestions on this manuscript, and Dr J. R. Menninger, The University of Iowa, Iowa City, USA for kindly providing us with the E.coli strains (AA7852, CP78) used in this study. We thank Mr Rahul Gaur for generating E.coli prfC::Kn strain. This work was supported by research grants from the Department of Science and Technology, the Department of Biotechnology and the Indian Council of Medical Research, New Delhi. N. Sadananda Singh is supported by a senior research fellowship of Council of Scientific and Industrial Research, New Delhi, India.
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* To whom correspondence should be addressed. Tel: +91 80 2293 2686; Fax: +91 80 2360 2697; Email: varshney@mcbl.iisc.ernet.in
ABSTRACT
The bacterial ssrA gene codes for a dual function RNA, tmRNA, which possesses tRNA-like and mRNA-like regions. The tmRNA appends an oligopeptide tag to the polypeptide on the P-site tRNA by a trans-translation process that rescues ribosomes stalled on the mRNAs and targets the aberrant protein for degradation. In cells, processing of the stalled ribosomes is also pioneered by drop-off of peptidyl-tRNAs. The ester bond linking the peptide to tRNA is hydrolyzed by peptidyl-tRNA hydrolase (Pth), an essential enzyme, which releases the tRNA and the aberrant peptide. As the trans-translation mechanism utilizes the peptidyl-transferase activity of the stalled ribosomes to free the tRNA (as opposed to peptidyl-tRNA drop-off), the need for Pth to recycle such tRNAs is bypassed. Thus, we hypothesized that tmRNA may rescue a defect in Pth. Here, we show that overexpression of tmRNA rescues the temperature-sensitive phenotype of Escherichia coli (pthts). Conversely, a null mutation in ssrA enhances the temperature-sensitive phenotype of the pthts strain. Consistent with our hypothesis, overexpression of tmRNA results in decreased accumulation of peptidyl-tRNA in E.coli. Furthermore, overproduction of tmRNA in E.coli strains deficient in ribosome recycling factor and/or lacking the release factor 3 enhances the rescue of pthts strains. We discuss the physiological relevance of these observations to highlight a major role of tmRNA in decreasing cellular peptidyl-tRNA load.
INTRODUCTION
For various physiological reasons, not all ribosomes that begin to translate an mRNA reach the termination codon. A significant population of the translating ribosomes stalls in between the decoding cycles (1,2). Stalled ribosomes pose a serious impediment to mRNA translation as they deplete pools of free tRNA and ribosomes, bringing protein synthesis to a halt. One of the cellular strategies to process stalled ribosomes involves peptidyl-tRNA drop-off (Figure 1, upper) (3–5). While the mechanisms responsible for the drop-off of peptidyl-tRNAs from the ribosomes are not very well understood (6), genetic and biochemical evidence (7–10) suggests that in E.coli, ribosome recycling factor (RRF) and EFG, as well as other factors such as RF3, IF1, IF2 and RelA contribute to this phenomenon. The peptidyl-tRNAs are recycled by peptidyl-tRNA hydrolase (Pth), which hydrolyzes the ester link between the tRNA and the peptide (11). As accumulation of peptidyl-tRNAs leads to cellular toxicity, Pth is an essential protein in bacteria (12,13).
Figure 1. Pathways depicting release of the tRNAs from the peptidyl-tRNAs by processing of stalled ribosomes pioneered either by dissociation of peptidyl-tRNAs (upper) or by tmRNA-mediated trans-translation (lower). Stalled ribosomes for trans-translation may arise either by endonucleolytic cleavage of mRNA in stalled ribosomes or from the translation of the truncated mRNAs.
Another cellular strategy that leads to the rescue of ribosomes stalled on fragmented mRNAs, involves trans-translation (14). Here the product of ssrA gene, tmRNA (also known as SsrA), is aminoacylated by alanyl-tRNA synthetase, and recruited to the ribosomal A site with the help of EFTu and an ancillary protein, SmpB. The peptidyl-transferase activity of the ribosome transfers the peptide from the P-site bound peptidyl-tRNA to the alanine on the –CCA end of the tmRNA. Consequently, the tRNA that was sequestered as peptidyl-tRNA in the stalled complex is released (15). The elongation process then resumes to translate a 30 nt long open reading frame (mRNA portion) of the tmRNA to encode the ANDENYALAA sequence tag (in E.coli). When the ribosomes reach the termination codon in the tmRNA, the C-terminally tagged peptide is released by the activity of release factors, and subjected to degradation by cellular proteases (16–18). Interestingly, as the tmRNA-mediated mechanism utilizes the peptidyl-transferase activity of the stalled ribosomes to liberate tRNA (as opposed to peptidyl-tRNA drop-off), the need for Pth to recycle such tRNAs is bypassed (Figure 1, lower). Since, a number of cellular processes result in accumulation of stalled ribosomes with fragmented mRNA (19–23), trans-translation may represent a major mechanism to recycle both the stalled ribosomes and the tRNAs. This raises an important question as to whether excess tmRNA in E.coli can alleviate the requirement for Pth activity.
Earlier studies have shown that RRF and RF3 are involved in peptidyl-tRNA drop-off from the ribosome. Thus, conditional lethal strains of E.coli, with a temperature-sensitive phenotype because of a mutation in pth could be rescued by downregulation of RRF expression (8). Conversely, overproduction of RRF in E.coli is toxic under conditions of limiting Pth activity (7). It was also observed that disruption of the prfC gene coding for RF3 resulted in suppression of the temperature-sensitive phenotype of a pthts strain. In vitro analyses suggested that disruption of prfC and downregulation of RRF result in decreased levels of peptidyl-tRNA drop-off (8). Also, the tRNALys appears to be one of the first species of tRNAs that is depleted by its sequestration as peptidyl-tRNA in the pthts strains of E.coli. Overproduction of this tRNA increases the temperature threshold at which the strains continue to grow (4,24).
Here, we show that overproduction of tmRNA rescues the temperature-sensitive phenotype of a pthts strain and that a major cellular function of tmRNA is to lessen the peptidyl-tRNA load. Further, coupling of tmRNA overproduction with downregulation of RRF and depletion of RF3, results in a stronger suppression of pthts. However, the role of Pth in E.coli is central and it was not possible to dispense with pth function even upon integration of the known suppressors in a single strain.
MATERIALS AND METHODS
Plasmids, strains and growth conditions
Plasmids and strains used in this study are listed in Table 1. Luria–Bertani (LB) liquid or solid (with 1.5% agar) media (Difco, USA) were used for growth (25). The medium was supplemented with various antibiotics at the following final concentrations: tetracycline, 7.5 μg/ml; kanamycin, 25 μg/ml; chloramphenicol, 30 μg/ml; and ampicillin, 100 μg/ml as required.
Table 1. List of plasmids/strains
Cloning
All constructs were made using standard recombinant DNA techniques (25) as follows:
E.coli ssrA gene
DNA primers 5'-GAAAAGCTTATTGGCTATCAC-3' and 5'-CACTAAGCTTGATTAACAGAG-3' containing HindIII sites were used to amplify the ssrA gene (along with its promoter) from E.coli HB101 DNA using Pfu DNA polymerase by PCR consisting of 30 cycles of incubations at 95°C for 1 min, 40°C for 30 s and 72°C for 2 min, digested with HindIII and cloned between the same sites of pTrc99C.
E.coli smpB-ssrA
The linked smpB and ssrA genes (along with their promoters) were PCR amplified from E.coli HB101 DNA by Pfu DNA polymerase using 5'-AACGGATCCCGGCGCTGGGT-3', and 5'-CACTAAGCTTGATTAACAGAG-3' primers containing BamHI and HindIII sites, respectively, by 30 cycles of incubations at 95°C for 1 min, 45°C for 30 s and 72°C for 2 min, digested with BamHI and HindIII, and cloned into similarly digested pTrc99C.
E.coli valU operon and generation of pTrc-valU-ssrA construct
The valU operon containing tRNALys gene was cloned from E.coli HB101 DNA by PCR using Pfu DNA polymerase with 5'-GCGAATTCTTTAGAAAATGC-3' and 5'-CAACTGGATCCTGCTGCAGGTTGG-3' primers, involving 30 cycles of incubations at 95°C for 1 min, 50°C for 30 s and 72°C for 1 min 20 s. The PCR product was digested with EcoRI and BamHI, and cloned between the same sites of pTrc-ssrA to obtain pTrc-valU-ssrA construct. The pTrc-valU construct was generated by deleting ssrA gene from the pTrc-valU-ssrA by digestion with HindIII followed by religation with T4 DNA ligase. The veracity of the clone was established by its ability to complement E.coli AA7852 for its pthts phenotype.
E.coli and Bacillus subtilis pth genes
The E.coli pth gene along with its promoter was amplified from E.coli HB101 using 5'-CACGTCTTGAAGGCAAACGG-3' and 5'-CGAACAACGTGACCGATCG-3' primers, digested with EcoRI and EcoRV, and cloned into the same sites of pTrc99C. Similarly, the open reading frame of B.subtilis pth gene was PCR amplified from B.subtilis genomic DNA using 5'-CGCCATGGTTGATTGCCGGTC-3' and 5'-CATTGAAGCTTCGAGACTGCC-3' primers. The PCR reaction involved 30 cycles of heating at 94°C for 1 min, 50°C for 45 s and 70°C for 1 min 45 s. The PCR product was digested with NcoI and HindIII, and cloned between the same sites of pTrc99C and pACDH.
Generation of E.coli strains
All strains described below were generated following the method of Datsenko and Wanner (30).
E.coli AA7852ssrA::Kn and E.coli CP78ssrA::Kn strains
pTrc-smpB-ssrA was digested with EcoRV and religated to delete a small fragment containing unwanted sequences of the plasmid and smpB origin to generate pTrc-smpB-ssrA*. This was digested with PvuII to cleave within the ssrA gene for cloning of a HincII DNA fragment carrying KnR cassette from pUC4K. This construct (pTrc-ssrA::Kn) was digested with EcoRV–HincII to obtain a 2.1 kb DNA containing the disrupted ssrA gene for electroporation into E.coli DY330. The ssrA disruption in E.coli DY330 was confirmed by PCR using flanking primers. The ssrA::Kn locus from the DY330 strain was mobilized into E.coli AA7852 (pthts) and E.coli CP78 (pth+, parent strain of AA7852) by P1-mediated transductions (31).
E.coli AA7852 prfC::Kn, E.coli CP79 prfC::Kn and E.coli CP79 prfC strains
The KnR cassette from pKD4 was amplified with 5'-CGCGGCGCGATGCCGCTTACTCAAGAAAGAATTGTGTAGGCTGGAGCTGCTTCG-3' and 5'-CAGGGCGCAGCGCGTACCGCGCCCTGGAGGCAAGAACATATGAATATCCTCCTTA-3' primers using Pfu DNA polymerase and electroporated into E.coli TG1 harboring pKD46. Transformants were screened for replacement of prfC with KnR cassette by PCR with the flanking primers (5'-GTCGAGCCAGGTAAACTC-3' and 5'-GCGACGGCAGAGGTCAAC-3') followed by digestion with PstI. The prfC::Kn was subsequently mobilized into E.coli AA7852 and E.coli CP79 by P1-mediated transductions to generate E.coli AA7852 prfC::Kn and E.coli CP79 prfC::Kn strains. The E.coli AA7852 prfC and E.coli CP79 prfC strains were obtained by excising the KnR cassette in the presence of pCP20 expressing the FLP recombinase (30,32).
E.coli AA7852 frr1 and E.coli CP79 frr1 strains
The strain (frr1) wherein RRF expression was down regulated was generated by insertion of a CmR cassette at the –32 position of frr promoter. The CmR cassette was PCR amplified from pKD3 using 5'-GGCAAATAAAATAGCCTAATAATCCAGACGATTACCCGGTGTAGGCTGGAGCTGCTTC-3' and 5'-GGAAGTGTGCTAAGTATAGCCCTGATTAAACATATTACATATGAATATCCTCCTTA-3' primers and electroporated into E.coli DY330. Integration of the CmR cassette was verified by PCR using flanking primers (5'-CGCAGCAACCGG-AGCGTAC-3' and 5'-GTTTCGCACTCGCTTCATCG-3'). E.coli AA7852 frr1 and E.coli CP79 frr1 strains were generated by P1-mediated transductions.
E.coli CP79 prfC, frr1
This strain was generated by P1-mediated transduction of frr1 locus (CmR) from AA7852frr1 to CP79prfC.
Generation of E.coli pth::kan
The KnR cassette was amplified from pUC4K using 5'-CTCCGCTGCGCGAAGAGGCTAAATTCTTTGGTTATACAAAGCCACGTTGTGTCTC-3' and 5'-CAGATCCAGTTCGTCGTGGGCCACCAGAATTTCGTCCGCGCTGAGGTCTGCCTCGTG-3' primers and electroporated into E.coli DY330 harboring pACDBsuPth. Transformants (KnR) were checked for disruption of the pth gene by PCR using flanking primers (5'-GTACGGATCCACGATTAAATTGATTGTCGGCCTG-3' and 5'-AGACAAGCTTTTATTGCGCTTTAAAGGCGTGCAA-3').
Analysis of SsrA expression
Total RNA was prepared from E.coli (33), electrophoresed on a 1.5% agarose gel and vacuum blotted to nytran membrane (25). The blot was probed with 5' 32P-labeled DNA oligomer (5'-GTTTCGCAAATCCCGTCGAAT-3') complementary to SsrA. The same blot was then probed for a second time with 5' 32P-labeled DNA oligomer (5'-TACCATCGGCGCTACGGCGTTTC-3') complementary to 5S rRNA (25). Blots were subjected to BioImage analysis (FLA2000, Fuji).
Analysis of peptidyl-tRNAs
Luria–Bertani medium (5 ml) containing ampicillin was inoculated with 2% inoculum from overnight cultures and grown at 30°C to an OD of 0.6 (595 nm), and shifted to 37°C for 45 min. The cells were then chilled on wet ice. All the subsequent steps for the isolation of total tRNA under acidic conditions at 4°C, separation on acid urea gels and electroblotting onto nytran membrane were as described in (34). The blot was probed (7) with a 5' 32P-labeled DNA complementary to tRNAHis, and tRNATyr (5'-GACAACTGGAATCACAATCC-3' and 5'-TACAGTCTGCTCCCTTTGGCCGCTC-3', respectively) and subjected to BioImage analysis (FLA2000, Fuji) using storage phosphor imager plates (BAS SR, Fuji).
Immunoblotting
The cell-free extracts from log-phase cultures were separated on 15% SDS–PAGE, transferred to PVDF membrane and probed with anti-RRF antibodies (7).
Analysis of essentiality of pth
Phage P1 lysate was raised on E.coli DY330 (pth::Kn) harboring pACDBsuPth and used to transduce E.coli CP79prfC, CP79frr1, and CP79prfC, frr1 harboring pTrc99C, pTrcBsuPth, pTrc-ssrA or pTrc-valU-ssrA. The transductants (KnR) were screened by PCR for pth locus with the primers (5'-CACGTCTTGAAGGCAAACGG-3' and 5'-CGAACAACGTGACCGATCG-3').
RESULTS
Overproduction of tmRNA (SsrA) rescues the temperature-sensitive phenotype of E.coli AA7852 (pthts)
To test the hypothesis if overproduction of tmRNA could rescue the pthts phenotype of E.coli AA7852, we introduced into this strain, various plasmid constructs (Table 1) by genetic transformation and checked for the growth of the transformants at different temperatures. As shown in Figure 2A, true to its genotype, the strain AA7852 harboring the pTrc99C vector alone grew at the permissive and semi-nonpermissive temperatures of 30 and 37°C, respectively but ceased to grow as the temperature was raised to 40°C (sectors 2). As a control, when the strain harbored a plasmid-borne copy of E.coli pth gene (pTrcEcoPth), it grew at 40°C (sectors 1). Interestingly, a plasmid-borne copy of ssrA gene (sectors 3) also rescued the growth of AA7852 at 40°C (compare sectors 2 and 3). Simultaneous presence of the extrachromosomal copy of smpB gene (coding for SmpB protein) along with ssrA (pTrc-ssrA-smpB) conferred no apparent advantage towards rescue by plasmid-borne ssrA (compare sectors 3 and 4), and was not analyzed any further. To examine the extent of tmRNA overproduction in the transformants, we analyzed the levels of tmRNA and 5S rRNA . Based on the relative levels of tmRNA and 5S rRNA , in the transformants harboring plasmid-borne copies of ssrA (pTrc-ssrA or pTrc-ssrA-smpB), production of tmRNA was about 4.5-fold higher than the chromosomal background suggesting that a moderate level of overproduction of tmRNA lessens the severity of the pthts phenotype in E.coli.
Figure 2. (A) Analysis of complementation of E.coli AA7852 (pthts) with ssrA. Various plasmids were introduced into E.coli AA7852 and the transformants were streaked on LB agar plates containing ampicillin, at various temperatures indicated above the plates. Sectors: 1, pTrcEcoPth; 2, pTrc99C (vector); 3, pTrc-ssrA; and 4, pTrc-ssrA-smpB. (B) Expression of tmRNA (SsrA) in E.coli AA7852 harboring different plasmids indicated above the lanes (or a ssrA::Kn null strain) was detected by northern blotting using radiolabeled DNA probes for tmRNA and 5 S rRNA . Relative levels of tmRNA (tmRNA/5S rRNA) shown in panel iii were obtained by quantification of signals by BioImage analyzer. Relative levels of tmRNA were normalized to transformants harboring pTrc99C vector (which was set as 1).
E.coli AA7852 (pthts) with ssrA::Kn allele are hypersensitive to temperature
In another approach to demonstrate that the chromosomally encoded tmRNA contributes to rescue of the pthts phenotype, we disrupted the ssrA gene in the E.coli AA7852 with Kn marker to generate E.coli AA7852ssrA::Kn strain. This procedure resulted in the complete absence of tmRNA in the AA7852ssrA::Kn strain . As seen in Figure 3A (sectors 3), the AA7852ssrA::Kn strain became hypersensitive to temperature and was not viable at 37°C, a temperature at which the parent strain AA7852 sustained good growth (Figure 2A, sector 2). Introduction of the plasmid-borne copy of ssrA into the AA7852ssrA::Kn strain, rescued its growth not only at 37°C but also at 40°C (Figure 3A, sectors 4) ruling out that lack of growth of AA7852ssrA::Kn strain at the semi-nonpermissive temperature was due to some non-specific effects emanating from the alterations at the chromosomal level. Further, as shown in Figure 3B, in liquid cultures also, while at 30°C, the AA7852ssrA::Kn strain harboring the vector alone, a plasmid-borne copy of ssrA or pth genes grew with similar rates; at 39°C, the AA7852ssrA::Kn strain harboring vector alone failed to show any significant growth. However, the growth of the strain at 39°C was rescued by the presence of a plasmid-borne copy of ssrA or pth. Expectedly, the rescue by pth was better than that by ssrA. In a control experiment, when the ssrA locus was disrupted in E.coli CP78 (pth+ isogenic strain of AA7852) in exactly the same manner, under the same conditions, no adverse effects of ssrA disruption were noticed (Figure 3A, compare sectors 1 and 2). Taken together, the observations in Figures 2 and 3 clearly suggest that ssrA is a suppressor of pthts phenotype in E.coli.
Figure 3. (A) Analysis of growth of E.coli CP78 (pth+, ssrA::Kn) harboring pTrc99C or pTrc-ssrA (sectors 1 and 2, respectively) and E.coli AA7852 (pthts, ssrA::Kn) harboring pTrc99C or pTrc-ssrA (sectors 3 and 4, respectively) on LB plates containing ampicillin at various temperatures indicated above the plates. (B) Analysis of growth in LB medium. Overnight cultures (0.05%) were inoculated into fresh medium and grown under shaking conditions at 30 or 39°C. Progress in growth was monitored by taking absorbance of samples at 595 nm at different times.
Overexpression of tmRNA lowers the steady-state levels of peptidyl-tRNAs in E.coli AA7852
To understand the mechanism of tmRNA-mediated rescue of the pthts phenotype, we analyzed the effect of its overexpression on the levels of peptidyl-tRNAs (peptidyl-tRNAHis and peptidyl-tRNATyr) in the cells. To ensure preservation of the sensitive ester bond that links amino acid or the peptide to the tRNA, RNA was prepared under cold and acidic conditions, and analyzed using acid urea gels. In these gels, tRNAs attached to peptides of heterogeneous length migrate slower than the aminoacylated tRNA, often in the form of a smear (7). Further, since the absolute levels of total tRNA vary in the preparations, to compare changes in the steady-state levels of peptidyl-tRNAs, their abundance as percent fraction of total tRNA (for tRNAHis or tRNATyr) was determined within each sample. As shown in Figure 4A, expectedly, at the permissive temperature of 30°C where the Pth is active, none of the transformants accumulate any significant levels of peptidyl-tRNAHis (lanes 1, 3 and 5). However, at the semi-nonpermissive temperature of 37°C, while the transformant harboring vector alone accumulated 54% of the tRNAHis as peptidyl-tRNAHis (lane 4), the presence of ssrA construct in the strain decreased this to 37% (lane 6). Expressing a wild-type copy of pth in AA7852, reduced the extent of accumulation of peptidyl-tRNAHis at 37°C to 11% (lane 2). Similar results were obtained with tRNATyr (Figure 4B). At 37°C, accumulation of tRNATyr as peptidyl-tRNATyr was 32% in transformants harboring vector alone (lane 4) which was decreased to 20% in transformants harboring the ssrA gene (lane 6). The level of peptidyl-tRNATyr in the transformants with plasmid-borne copy of wild-type pth at 37°C, was 9% (lane 2). Thus, consistent with our hypothesis of ssrA serving as a suppressor of pthts, overproduction of tmRNA at 37°C resulted in decreased accumulation of both the tRNATyr and tRNAHis in the form of peptidyl-tRNAs.
Figure 4. Analysis of the accumulation of peptidyl-tRNAs in E.coli AA7852 (pthts) in the presence of vector (pTrc99C) or pTrc-ssrA. Transformants were grown at permissive (30°C) or semi-nonpermissive (37°C) temperatures. Total tRNA was prepared under acidic conditions, fractionated on acid-urea gel, transferred to nytran membrane and hybridized with 5' end 32P-labeled anti-tRNAHis (A) or anti-tRNATyr (B) probes. Signals were quantified by BioImage analyzer (Fuji) and % peptidyl-tRNA was calculated as . These are shown below the lanes.
ssrA-mediated suppression of pthts in prfC, and frr promoter down backgrounds
Heurgue-Hamard et al. (8) reported that disruption of prfC (RF3), or the promoter region of frr (RRF) resulted in suppression of pthts phenotype in E.coli. In order to integrate these results and the tmRNA effect described above, and to further our understanding on the mechanisms that lead to suppression of the pthts phenotype, we generated a prfC derivative of AA7852 by replacement of the open reading frame of RF3 with KnR marker, and a promoter down frr by insertion of CmR marker at –32 position in the promoter region (frr1). Genetic manipulations in both strains were confirmed by PCR analysis (Materials and Methods). Further, an immunoblot analysis of the cell-free extracts obtained from the frr1 derivative showed downregulation of expression of RRF . Yet another derivative of AA7852 that we generated possessed both the prfC and frr1 genetic alterations. As expected, when compared with the parent strain AA7852 (Figure 6, sector 2), all the three derivatives of AA7852 (prfC, frr1 and the double mutation of prfC and frr1) rescued the phenotype of the pthts strain (sectors 4, 6 and 8, respectively, at 40 and 42°C). More importantly, introduction of a plasmid-borne ssrA gene in all of these strains resulted in a higher efficiency of suppression of pthts in AA7852 (compare sectors 4, 6 and 8 with 3, 5 and 7, respectively at 42°C). At 42°C, it is seen that rescue by prfC and frr1 markers is somewhat better than that conferred by overproduction of tmRNA (compare sectors 1 with 4 and 6, respectively). Nevertheless, integration of prfC, frr1 with a plasmid-borne copy of ssrA (sector 7) resulted in a remarkable rescue of E.coli AA7852.
Figure 5. Immunoblot analysis of total cell-free extracts of E.coli AA7852 (lane 2) and its frr1 derivative (lane 3) for RRF. Cell-free extracts (10 μg) were analyzed by SDS–PAGE and detected by Coomassie blue stain . A similar gel was prepared for transfer to PVDF membrane and immunoblotted using anti-RRF antibodies . Lane 1 contains pure RRF as marker.
Figure 6. Analysis of growth of E.coli AA7852 (sectors 1 and 2), and its derivatives E.coli AA7852prfC::Kn (sectors 3 and 4), E.coli AA7852frr1 (sectors 5 and 6), and E.coli AA7852frr1, prfC::Kn (sectors 7 and 8) harboring pTrc99C (sectors 2, 4, 6 and 8) or pTrc-ssrA (sectors 1, 3, 5 and 7) at various temperatures as indicated on LB agar plates containing ampicillin.
pth is essential in E.coli
With the identification of tmRNA as an additional suppressor of pth, there are now at least five factors (RRF, RF3, RelA, tRNALys and tmRNA) whose influence on Pth is well characterized in E.coli. Of these, RF3, RRF and RelA are responsible for increased drop-off of peptidyl-tRNA, and tmRNA leads to a decrease in the level of peptidyl-tRNA. On the other hand, tRNALys (in valU operon) functions at a step downstream of the peptidyl-tRNA release. These observations, together with the fact that both of the identifiable pth genes in Saccharomyces cerevisiae could be deleted, raise the question of whether the pth gene in E.coli can be dispensed with. To address this, we first integrated one or many of the conditions that lead to rescue of pthts, into E.coli CP79 (relA–). We also generated an E.coli strain wherein 0.1 kb segment of the chromosomal pth open reading frame was replaced with a kanamycin resistance marker (pth::Kn) in the presence of a helper plasmid harboring a pth gene of B.subtilis origin under the control of an E.coli promoter (Figure 7A). Phage P1 lysate was raised on this strain to attempt knockout of the pth gene in the E.coli CP79 derivatives by transduction. As shown in Table 2, a number of 1000 transductants on a Kn plate with a control strain harboring plasmid-borne pth gene of B.subtilis origin suggests a high efficiency of tranduction. However, despite many attempts, the number of transductants on the test strains, which lacked a pth support plasmid but into which various suppressors of pth had been integrated, was very low (Table 2). Further, PCR screening (Figure 7A) of the entire test transductants obtained, revealed that they either possessed an intact copy, or both an intact and a disrupted copy of the pth genes in their chromosomes (a representative gel is shown in Figure 7B, and data are summarized in Table 2). On the other hand, the control transductants (obtained in the presence of support plasmid) possessed only a disrupted copy of the E.coli pth gene (Figure 7B, lane 1). Use of several other strains of E.coli in such experiments yielded identical results (data not shown). Taken together, these observations suggest that pth plays a central role and is indispensable in E.coli.
Figure 7. Analysis of pth gene disruption in E.coli. (A) Schematic representation of the pth locus and its disruption by Kn cassette in E.coli. Expected sizes of the PCR products from chromosomal DNAs of an intact and disrupted loci are as indicated. (B) PCR screening of representative transductants. Lanes: M, DNA size markers ( DNA digested with HindII and HindIII); WT, wild-type locus; lane 1, positive control transductant (disrupted locus); lanes 2–7, transductants obtained on CP79prfC, frr1, pTrc-ssrA (lanes 2 and 3), CP79 frr1, pTrc-ssrA (lanes 4 and 5) and CP79prfC, frr1, pTrc-valU-ssrA (lanes 6 and 7) backgrounds.
Table 2. Analysis of pth gene disruption in E.coli
DISCUSSION
In bacteria, the product of ssrA gene, SsrA is a well-conserved molecule of 360 nt in length. In E.coli, SsrA was originally identified as 10Sa RNA upon further fractionation of the 10S RNA (35). Subsequently, SsrA was shown to function both as tRNA and mRNA, hence the new name tmRNA (14). The 3' end of tmRNA terminates with a –CCA sequence to which alanine is ligated by alanyl-tRNA synthetase. The alanylated-tmRNA is recruited to the ribosomal A-sites of the stalled ribosomes for the process of trans-translation, which attaches an AANDENYALAA sequence (in E.coli) to the C-terminal of the incomplete peptides and directs the aberrant proteins for degradation by the cellular proteases (4,36–38). In this study, we have uncovered yet another facet of tmRNA function in decreasing the peptidyl-tRNA load in the cells.
Ribosomes may stall on either a full-length or a truncated mRNA. While the stalled complexes with truncated mRNAs (with no or very short 3' extensions) are direct and preferred substrates for tmRNA, even those with an intact mRNA may be directed to trans-translation following the action of codon-specific ribonuclease (19–22). The observation that in the wild-type ssrA background, overproduction of tmRNA raises the threshold of the temperature sensitivity of the pthts strain suggests that in the cells, there exists a population of stalled ribosomes, which is the substrate for trans-translation, but remains unprocessed by the available tmRNA. Consequently, this population of stalled ribosomes (which may possess either a truncated or intact mRNA) must be processed via alternate pathways resulting in dissociation of peptidyl-tRNAs from them, which in turn should be processed by Pth to recycle the tRNAs. Thus, we believe that rescue of the pthts strain AA7852, by overproduction of tmRNA is due to diversion of this population of the stalled ribosomes from dropping-off peptidyl-tRNAs to engaging them in trans-translation (Figure 1, lower). This will in turn, decrease the substrate load on Pth, and make the available Pth activity sufficient to process peptidyl-tRNAs that arise from other physiological processes. Further, although the exact mechanisms by which RRF and RF3 result in peptidyl-tRNA drop-off are not understood, it is clear that downregulation of RRF and/or absence of RF3 result in decreased peptidyl-tRNA load in the cell (7,8). Interestingly, as shown in Figure 6, overproduction of tmRNA in a strain harboring frr1 and prfC mutations in single or in combination, results in a synergistic effect on the rescue of the pthts phenotype. The synergism between the two mutations themselves and with ssrA suggests that the stalled ribosomes are channeled to utilization by all the three factors and, while the RRF and RF3 pathways rescue them by drop-off of peptidyl-tRNAs (Figure 1, upper), ssrA rescues them by trans-translation (Figure 1, lower).
Studies on mutants of E.coli and its phages have unraveled crucial aspects of the host's physiology (39). It was observed that rap (same as pth) mutation in E.coli resulted in defective growth of bacteriophage (40,41). Interestingly, yet another independent study, reported a similar phenotype for the sip (same as ssrA) mutation in E.coli (42,43). While no link has been established between these two separate studies, these observations support the importance of ssrA and the physiological relevance of the connection between ssrA and pth that we have established in this study. In fact, at least in Neisseria gonorrhoeae, the ssrA is an essential gene (44). It will be interesting to investigate the status of peptidyl-tRNA/Pth in this organism.
The genetic and biochemical analyses of the suppressor mutations (bar mutations) in phage that rescue the growth on rap (pth) mutants of E.coli have shown that the phage-encoded minigenes (wild-type bar regions, at least one of which is located in the imm region) are responsible for excess drop-off of the peptidyl-tRNAs and depleting the availability of tRNA for translation (45). Such peptidyl-tRNAs are recycled by Pth. Hence, the effect of Pth on phage growth is a direct one. Whether the effect of ssrA on phage growth is also a direct one (e.g. by trans-translation) is not clear. At least, the lack of tmRNA-mediated targeting of aberrant proteins to degradation is not responsible for the bacteriophage growth defect in sip (ssrA) mutants (38). On the other hand, based on recent reports that the ribosomes with truncated mRNAs with no or very short 3' extensions are the real substrates for trans-translation by tmRNA (46,47) and that the minigene toxicity is more a consequence of frequent re-initiation events (48), it appears that the ssrA effect on bacteriophage growth is an indirect one. The presence of ssrA most likely decreases peptidyl-tRNA load by engaging the stalled ribosomes into trans-translation (thereby withdrawing the chances of the factor-dependent or spontaneous processes that result in drop-off of peptidyl-tRNAs). This process, frees Pth to now hydrolyze the peptidyl-tRNAs arising out of the minigenes of the bacteriophage , or other host activities. However, this interpretation does not rule out a direct role for ssrA in the bacteriophage growth. Recently, it has been shown that at least within certain codon contexts, much of the peptidyl-tRNA population arising from minigene expression remained in the ribosomes (49). Such ‘stand-by’ stalled ribosomal complexes can be further processed to become substrates for trans-translation (19–22). Therefore, it will be interesting to study the mechanism by which ssrA contributes to the lambdoid phage growth.
Finally, biochemical studies show that Saccharomyces cerevisiae possesses at least two distinct types of Pth activities similar to those found in bacteria and archaea. Recently, it was shown that deletions in S.cerevisiae of the two identifiable pth genes (individually or in combination) were viable (50,51). A large number of factors in E.coli (RRF, RF3, IF1, IF2, EFG, tmRNA, RelA, tRNALys, etc.) affect peptidyl-tRNA levels in cell. And, the fact that many of these have also been characterized as suppressors of Pth suggests that the role of Pth in E.coli (and possibly in other organisms) is central to the process of mRNA translation. On the other hand, the knowledge of a large number of suppressors that decrease the peptidyl-tRNA load in E.coli also makes it desirable to attempt to delete the only known pth gene in E.coli. Thus, we integrated many of the known suppressor markers in E.coli to attempt deletion of pth gene. However, our studies (Figure 7) clearly support the fact that the Pth activity is indeed an essential function. In fact, as has also been suggested by the authors of the S.cerevisiae study (51), there may well be several Pth-like activities in eukaryotes to recycle tRNAs. In rabbit reticulocyte lysates, one of the activities that recycles tRNAs from the peptidyl-tRNAs is a phosphodiesterase that cleaves between the C and A, the last two nucleotides in the tRNA (52). The tRNA is subsequently repaired by tRNA nucleotidyltransferase (53). Whether such Pth activity is present in yeast is not known. Certainly, the reaffirmation of the essentiality of Pth activity in E.coli is a good indicator of the presence of additional Pth activities in yeast.
ACKNOWLEDGEMENTS
We thank Dr U. L. RajBhandary and our laboratory colleagues for their suggestions on this manuscript, and Dr J. R. Menninger, The University of Iowa, Iowa City, USA for kindly providing us with the E.coli strains (AA7852, CP78) used in this study. We thank Mr Rahul Gaur for generating E.coli prfC::Kn strain. This work was supported by research grants from the Department of Science and Technology, the Department of Biotechnology and the Indian Council of Medical Research, New Delhi. N. Sadananda Singh is supported by a senior research fellowship of Council of Scientific and Industrial Research, New Delhi, India.
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