A physiological connection between tmRNA and peptidyl-tRNA hydrolase functions in Escherichia coli

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 (pth(ts)). Conversely, a null mutation in ssrA enhances the temperature-sensitive phenotype of the pth(ts) 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 pth(ts) strains. We discuss the physiological relevance of these observations to highlight a major role of tmRNA in decreasing cellular peptidyl-tRNA load.

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.

Figure 2

(A) Analysis of complementation of E.coli AA7852 (pth^ts) 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 [panel (i)] and 5 S rRNA [panel (ii)]. 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).

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 (pth^ts, 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.

Figure 4

Analysis of the accumulation of peptidyl-tRNAs in E.coli AA7852 (pth^ts) 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-tRNA^His (A) or anti-tRNA^Tyr (B) probes. Signals were quantified by BioImage analyzer (Fuji) and % peptidyl-tRNA was calculated as [(peptidyl-tRNA/(peptidyl-tRNA + aminoacyl-tRNA + tRNA) × 100]. These are shown below the lanes.

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 [panel (i)]. A similar gel was prepared for transfer to PVDF membrane and immunoblotted using anti-RRF antibodies [panel (ii)]. 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 AA7852ΔprfC::Kn (sectors 3 and 4), E.coli AA7852frr1 (sectors 5 and 6), and E.coli AA7852frr1, ΔprfC::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.

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 (λ DNA digested with HindII and HindIII); WT, wild-type locus; lane 1, positive control transductant (disrupted locus); lanes 2–7, transductants obtained on CP79ΔprfC, frr1, pTrc-ssrA (lanes 2 and 3), CP79 frr1, pTrc-ssrA (lanes 4 and 5) and CP79ΔprfC, frr1, pTrc-valU-ssrA (lanes 6 and 7) backgrounds.