Translation initiation from the ribosomal P‐site is the specialty of the initiator tRNAs (tRNAfMet). Presence of the three consecutive G‐C base pairs (G29‐C41, G30‐C40 and G31‐C39) in their anticodon stems, a highly conserved feature of the initiator tRNAs across the three kingdoms of life, has been implicated in their preferential binding to the P‐site. How this feature is exploited by ribosomes has remained unclear. Using a genetic screen, we have isolated an Escherichia coli strain, carrying a G122D mutation in folD, which allows initiation with the tRNAfMet containing mutations in one, two or all the three G‐C base pairs. The strain shows a severe deficiency of methionine and S‐adenosylmethionine, and lacks nucleoside methylations in rRNA. Targeted mutations in the methyltransferase genes have revealed a connection between the rRNA modifications and the fundamental process of the initiator tRNA selection by the ribosome.
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.
Specific interactions between ribosome recycling factor (RRF) and elongation factor-G (EFG) mediate disassembly of post-termination ribosomal complexes for new rounds of initiation. The interactions between RRF and EFG are also important in peptidyl-tRNA release from stalled pre-termination complexes. Unlike the post-termination complexes (harboring deacylated tRNA), the pre-termination complexes (harboring peptidyl-tRNA) are not recycled by RRF and EFG in vitro, suggesting participation of additional factor(s) in the process. Using a combination of biochemical and genetic approaches, we show that, (i) Inclusion of IF3 with RRF and EFG results in recycling of the pre-termination complexes; (ii) IF3 overexpression in Escherichia coli LJ14 rescues its temperature sensitive phenotype for RRF; (iii) Transduction of infC135 (which encodes a functionally compromised IF3) in E.coli LJ14 generates a ‘synthetic severe’ phenotype; (iv) The infC135 and frr1 (containing an insertion in the RRF gene promoter) alleles synergistically rescue a temperature sensitive mutation in peptidyl-tRNA hydrolase in E.coli; and (v) IF3 facilitates ribosome recycling by Thermus thermophilus RRF and E.coli EFG in vivo and in vitro. These lines of evidence clearly demonstrate the physiological importance of IF3 in the overall mechanism of ribosome recycling in E.coli.
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