| dc.description.abstract | Translation is a basic process of the cell wherein the genetic code/message in the form of codons of mRNA is decoded into proteins — the functional entities of the cell. The process of translation is divided into four steps — Initiation, Elongation, Termination, and Recycling in prokaryotes. A deep understanding of this process in light of structural and biochemical data, with integration of genetics, is an objective of this study.
The work described in this thesis, entitled “The mechanism of decoding at the P-site of the ribosome and the role of 3 GC base pairs in initiator-targeting the initiator tRNA to the P-site of the ribosome in Escherichia coli”, is an attempt to uncover the finer details of the initiation step of translation using insights from structural and biochemical data on selection and passage of tRNA at the P-site of the ribosome (initiator and peptidyl-tRNA).
The thesis is divided into five chapters. Chapter 1 (Introduction) briefly describes the relevant published work, beginning with the details of the steps of the process of translation and introducing the unique features of the P-site of the ribosome, which form a framework to understand the questions asked in the study. The next chapter, Materials and Methods, provides technical details wherein the model organism/strains and experimental procedures used throughout the study are described and referred to in the rest of the chapters. The rest of the thesis is an amalgamation of outcomes of experiments done to answer the questions asked in the study, which are sectioned into three chapters as follows:
(i) Unravelling the mechanism of decoding at the P-site of the ribosome at the step of initiation
Initiation is the rate-limiting step of translation. Understanding initiation is key to resolving the puzzles of translation. A major limitation to understanding the process is the lack of a high-resolution crystal structure of the initiation complex. The P-site is where initiator tRNA is poised directly for translation and is uniquely endowed with post-transcriptionally modified nucleosides and tails of two proteins, S9 and S13.
The modified nucleosides have been implicated in the fidelity of translation in a previous study in our laboratory. The tails of the two proteins intruding into the P-site have been a subject of study using in vitro approaches since 2004 by Noller’s group and later by Fredrick’s group in 2009. We studied the in vivo relevance of two methylated nucleosides, G966 and C967, that lie in the vicinity of initiator tRNA, and of a conserved tripeptide sequence (SKR) in the S9 tail, which contacts initiator tRNA/P-site tRNA.
In this regard, we tested their role in the maintenance of initiation with the most efficient initiation codon AUG and with alternate codons GUG, UUG, and CUG, which are also efficiently utilized as initiation codons, and in suppressing initiation from non-canonical or rarely utilized initiation codons AUA, AUC, AUU, and ACG. We also tested if they can prevent non-initiator or unnatural tRNAs at the P-site by mutating initiator tRNA’s anticodon to various others and finally tested whether these features inspect the three consecutive GC base pairs of initiator tRNA.
We found that the contributions of these features varied quantitatively, with methylated nucleosides G966 and C967 having subtle effects. The C-terminus of S9 plays a major role in decoding, i.e., maintaining initiation with AUG, GUG, and UUG codons, thus maintaining the initiation site/5’ wobble (which differs from A-site/3’ wobble). It disfavors anticodon mutants of initiator tRNA and also inspects the P-site tRNA for the presence of three consecutive GC base pairs. Thus, we show that, like the A-site, the P-site also has an inherent mechanism of decoding that involves the tail of S9 protein and methylated nucleosides G966 and C967. This interpretation is further supported by molecular dynamics simulations carried out on various codon:anticodon interactions in the P-site.
(ii) S9 protein, G966, and C967 affect fidelity at initiation as well as elongation of translation
This chapter describes additional phenotypes of fidelity factors whose role in initiation was investigated in the previous chapter. In Chapter 3, initiation with alternate initiation codons — GUG, UUG, and CUG — was reduced upon deletion of the S9 tail. Molecular dynamics simulation revealed that initiator tRNA is held less tightly at the P-site upon deletion of the SKR motif of the S9 tail.
This prompted us to investigate the role of unique P-site features — S9 tail and methylated G966 and C967 nucleosides, independently and in combination — in gripping peptidyl-tRNA and maintaining the reading frame. Maintenance of the reading frame is critical for both initiation and elongation steps of translation.
Our results indicate that the tail of S9 protein is important in gripping peptidyl-tRNA and maintaining the reading frame. The methylated nucleosides independently do not contribute much to reading frame maintenance, but a combination of deletion of the S9 tail and rsmB (C967 methyltransferase) shows a higher level of -1 frameshift errors than deletion of the S9 tail alone. We also observed that a combination of some P-site fidelity factors (deletion of S9 and C967 methyltransferase gene, rsmB) reduced the fitness of strains at low temperatures.
In addition, we investigated the role of a modification, Q (which modulates frameshifting), on growth phenotype at different temperatures independently and in combination with the above-mentioned P-site features. Lack of Q modification shows a high level of frameshift errors in Escherichia coli strains, which is suppressed many folds by deletion of either of the two P-site fidelity factors — S9 tail or rsmD, or both. In future, polysome profiling of some of these strains should be done to reveal if these strains have a defective P-site and hence a biogenesis defect.
(iii) The role of 3 GC base pairs in initiator-elongator discrimination at the P-site of the ribosome in E. coli
The role of the conserved three GC base pairs of initiator tRNA in the initiation of translation and targeting the initiator tRNA to the P-site is of interest to the laboratory. This was analyzed using second-site suppressors that initiate with a 3 GC mutant initiator tRNA. Two such suppressors, B33 and B21, were studied.
The mutation in B33 mapped to the locus that contributes 80% of initiator tRNA to the cell. It is a promoter-down mutation that has already been characterized in the laboratory. It is a cold-sensitive strain, and a 2D proteome analysis was pursued at 37 °C and 25 °C to suggest how differences at the proteomic level may contribute to this phenotype.
Similarly, another suppressor, B21, which was already mapped to the 85’ locus, was also studied. Fine mapping and linkage analysis predicted the location of the mutation in one of the seven 16S rRNA genes at the 85’ locus. Sequencing of the 16S rRNA locus from the rrnB operon showed a mutation in the anti-Shine-Dalgarno sequence, making it more complementary to the Shine-Dalgarno sequence of our reporter gene, thus leading to initiation from the reporter. | |
