The studies reported in this thesis describe the work done in the area of translation initiation where a previously unknown role of multiple copies of initiator tRNA in E. coli has been reported. Also the role of SsrA resume codon in resumption of translation, until not clearly known has been reported here. Chapter -1 discusses the relevant literature in understanding translation and initiator tRNA selection on the ribosome during initiation. It also discusses the literature pertaining to the aspect of release of stalled ribosomal complexes by SsrA. This is followed by the next chapter (chapter- 2) which discusses the materials and methods used throughout the study. Chapter- 3 describes the studies leading to the role of multiple copies of initiator tRNA in E. coli in governing the fidelity of initiator tRNA selection on the P site of the ribosome. This is followed by Chapter-4 which describes the role of the resume codon of the SsrA in governing the efficiency of trans-translation in releasing the stalled ribosomal complexes. The summaries of the chapters 3 and chapter 4 are briefly described below. i) Role of conserved 3GC base pairs of initiator tRNA in the initiator-elongator tRNA discrimination. Translation initiation is the first step in the very important and highly conserved biological process of protein biosynthesis. The process involves many steps, a wide array of protein factors at each specialized step and a large ribonucleoprotein particle; the ribosome to decode the information of the mRNA template into biologically active proteins. The process of initiation is still unclear largely due to fewer reports of available structural data. One of the very interesting questions that people have been trying to address is how the initiator tRNA is selected on the P- site of the ribosome and what is the importance of the conserved three GC base pairs in the anticodon stem of the initiator tRNA. Here in this study, I have studied this question by using the classical genetic technique of generating and characterizing the mutant initiator tRNA defective at the step of initiation. I have identified and analyzed the suppressors which are capable of rescuing this defect in initiation. The study involves two such E. coli suppressor strains (named D4 and D27). These suppressors can initiate translation from a reporter CAT mRNA with amber codon, independent of the presence of the three consecutive GC base pairs in the anticodon stem of initiator tRNAs. Mapping of the mutations revealed that the mutants are defective in expression of the tRNA1fMet (metZVW) gene locus which encodes the initiator tRNA. Both the suppressors (D4 and D27) also allow initiation with elongator tRNA species in E. coli. Taken together, the results show that E. coli when deficient in the initiator tRNA concentration can lead to initiation with elongator tRNA species. ii) The Role of SsrA/tmRNA in ribosome recycling and rescue. Occasionally during the process of translation, the ribosomes stall on the mRNA before the polypeptide synthesis is complete. This situation is detrimental to the organism because of the sequestration of the tRNAs as ‘peptidyl tRNAs’ and the ribosomes. In E. coli one of the pathways to rescue stalled ribosomes involves disassembly of these stalled complexes to release peptidyl tRNAs which are then recycled by peptidyl tRNA hydrolase (Pth), an essiential enzyme in E. coli. The other pathway which is not essential in E. coli but is conserved in all prokaryotes involves SsrA or tmRNA (transfer messenger RNA). The tmRNA is charged with alanine and recognizes the stalled ribosomal complexes and acts as tRNA to bind the A-site. It also functions as mRNA by adding a undecapeptide (which is actually a tag for degradation by cellular proteases) to the existing polypeptide and there is normal resumption of the translation. In most sequences of SsrA ORF, the first codon of the ORF, called as resume codon, is conserved. I wanted to understand the importance of the conservation of the resume codon. Towards this end I randomly mutated the resume codon and studied the effect of the altered resume codon in the rescue of stalled ribosomal complexes. The effect of over-expression of these mutants was investigated in the rescue of the Pthts defect since it is known that the overexpression of SsrA rescues the temperature sensitive phenotype of the Pthts strain and so causes less accumulation of peptidyl–tRNA in E. coli .The effect for these mutants has also been studied by the growth of hybrid λimmP22 phages. I also used AGA minigene system to study the effect of various mutants which has been shown to sequester tRNAArg (UCU) in the ribosomal P-site, translation of this minigene causes toxicity to E. coli. I have tried to study the effect of the SsrA mutants in rescue of toxicity caused by the minigene. Overall, the observations indicate that the conservation of the resume codon is important in E. coli and having mutated resume codon probably leads to deficient trans-translation during one or the other growth conditions.

Originally 2A was characterised in foot-and-mouth disease virus. Site directed mutagenesis identified a C-terminus consensus motif [D(V/I)ExNPGP] and it is proposed that 2A interacts with the exit tunnel of the ribosome in a way that a specific peptide bond is skipped between the last glycine of 2A and the proline of 2B, thus providing a discontinuity in translation, resulting in release of discrete proteins from one single ORF. 2A was also identified in other picornaviruses, positive, single and double-stranded RNA insect viruses and mammalian rotaviruses. A motif present at the C-terminus of the 2A oligopeptide [D(V/I)ExNPGP] is very highly, though not completely conserved . The sequence upstream of this motif shows, however, no apparent conservation between 2As of different viruses. In this study, extensive site-directed mutagenesis were performed on several 2A sequences and a series of ‘hybrid' 2As comprising different consensus motifs juxtaposed with different upstream contexts were created as part of a detailed analysis of the mechanism of 2A-mediated ribosome stalling. The results demonstrated that a minimal region of twenty to twenty-three amino acids interacts with the exit tunnel of the ribosome to bring about a pause in processivity, alter the peptidyl transferase centre geometry and restrict the ribosome A site via two distinctive stalling mechanisms. Other molecular analyses tested here will require further optimisations or alternative methods: a visual method to explore the dynamics of re-initiation of translation from proline codon, purification of the translation-regulating factors and structural resolution of 2A sequences. Previously, cellular 2As were identified in non-LTR retrotransposons of trypanosomes. It is reported here as part of two other cellular organisms Saccoglossus kowalevskii (acorn worm) and Branchiostoma floridae (amphioxus). In the acorn worm, the nucleotides sequences corresponding to 2A motifs were part of the untranslated genome. In amphioxus, three 2A elements were identified in hypothetical proteins, and at the N-terminus of twenty non-LTR retrotransposons.

Studies reported in this thesis address the question of how pre-termination ribosomal complexes stalled during translation of mRNA are recycled. The process of recycling of the stalled ribosomes involves many translational factors. During the course of my studies, I have uncovered new roles of SsrA (tmRNA), IF3 and ribosome recycling factor (RRF) in recycling stalled ribosomes. These findings are summarized as follows: (i) A physiological connection between tmRNA and peptidyl-tRNA hydrolase functions in Escherichia coli 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 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. The findings of the experiments detailed in this thesis show that SsrA rescues a defect in Pth by reducing the peptidyl-tRNA load on Pth. (ii) Evidence for a role of initiation factor 3 in recycling ribosomal complexes stalled on mRNAs in Escherichia coli. Specific interactions between ribosome recycling factor (RRF) and EF-G mediate disassembly of post-termination ribosomal complexes for new rounds of initiation. The interactions between RRF and EF-G are also important in peptidyl-tRNA release from pre-termination complexes. Unlike the post-termination complexes (harboring tRNA), the pre-termination complexes (harboring peptidyl-tRNA) are not recycled by RRF and EF-G in vitro, suggesting participation of additional factor(s) in the process. Using a combination of biochemical and genetic approaches, we show that, 1. Inclusion of IF3 with RRF and EF-G results in recycling of the pre-termination complexes; 2. IF3 overexpression in Escherichia coli LJ14 rescues its temperature sensitive phenotype for RRF; (3) Transduction of infC135 (encoding functionally compromised IF3) in E. coli LJ14 generates a ‘synthetic severe’ phenotype; (4) The infC135 and frr1 (a promoter down RRF gene) alleles synergistically rescue a temperature sensitive mutation in peptidyl-tRNA hydrolase in E. coli; and (5) 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. (iii) The role of RRF in dissociating of pre-termination ribosomal complexes stalled during elongation Translating ribosomes often stall during the repetitive steps of elongation for various reasons. The stalled ribosomes are rescued by the process of trans-translation involving tmRNA (SsrA) or by a factor mediated dissociation of the stalled ribosome into its subunits leading to the drop-off of the peptidyl-tRNA. The mechanistic details of how the factor mediated dissociation is carried out, is not well studied. Studies described in the above section have highlighted the role of RRF in dissociating stalled pre-termination complexes. However, the in vivo studies in this area have been limited for lack of defined pre-termination complexes. Two in vivo systems based on translation of AGA minigene and the ung gene (EcoUngstopless) transcripts were designed. Evidence is presented to show that translation of both of these transcripts is toxic to E. coli because of the accumulation of the transcript specific stalled pre-termination complexes. Availability of these model systems has allowed us to address the role of RRF in dissociating stalled ribosomes. We show that RRF rescues stalled ribosomes on these constructs and its overexpression can rescue the toxicity. The physiological importance of this observation is highlighted by the rescue of AGA minigene inhibitory effect on λimmP22 hybrid phage growth upon RRF overexpression.