Mechanism Of Ribosome Recycling In Eubacteria, And The Impact Of rRNA Methylations On Ribosome Recycling And Fidelity Of Initiation In Esherichia coli
The studies reported in this thesis address, firstly, aspects of ribosome recycling in eubacteria, and secondly, a preliminary characterization of an EFG-like locus from Mycobacterium smegmatis. A hitherto unsuspected role of the ribosome recycling factor in governing the fidelity of initiation has been discovered during the course of this work. A summary of the relevant literature is presented in chapter 1. Section I of the ‘General Introduction’ provides a brief review of the current understanding of protein biosynthesis, with a special emphasis on ribosome recycling and the fidelity of translation initiation. Section II provides a brief introduction to mycobacterial translation, and known deviations from the E. coli prototype are highlighted. This is followed by three chapters containing experimental work, as summarized below. (i) Role of elongation factor G in governing specificity of ribosome recycling In eubacteria and the eukaryotic organelles, the post-termination ribosome complexes are recycled by the combined action of ribosome recycling factor (RRF) and elongation factor G (EFG). Earlier studies both from our laboratory and other laboratories have revealed the existence of specific interactions between RRF and EFG that are crucial for ribosome recycling, using ribosomes from E. coli and factors from both E. coli and heterologous sources such as Mycobacterium tuberculosis, Thermus thermophilus etc. In this study, to further understand the mechanism of ribosome recycling, we employed polysomes from both E. coli and M. smegmatis and monitored ribosome recycling in in vitro assays using RRF and EFG from both these sources; in addition, in vivo assays were performed in E. coli using either temperature-sensitive strains or strains carrying a deletion in frr (encoding RRF) or fusA (encoding EFG) genes. It was found that, in E. coli, RRF from Mycobacterium tuberculosis and M. smegmatis function with MtuEFG or MsmEFG but not with EcoEFG. In vitro assays revealed that the mycobacterial EFGs facilitate recycling of both the mycobacterial and E. coli polysomes not only with mycobacterial RRFs but also with EcoRRF. In contrast, although EcoEFG binds to mycobacterial polysomes, carries out GTP hydrolysis and is reported to sustain translocation on mycobacterial ribosomes, its activity in recycling mycobacterial polysomes was undetectable with EcoRRF, as well as with the mycobacterial RRFs. Such an observation allowed us to infer that EFG establishes specific interactions with the ribosome that are crucial for ribosome recycling but not for translocation, suggesting that translocation and ribosome recycling are distinct functions of EFG. In addition, a number of EFG chimeras generated by swapping corresponding domains between Msm- and Eco-EFGs were analyzed for their ability to sustain translocation and/or ribosome recycling in E. coli and M. smegmatis, using a combination of in vivo (for E. coli) and in vitro (for both E. coli and M. smegmatis) approaches. Our observations reveal that a dual set of specific interactions of EFG with RRF and ribosome is essential for ribosome recycling. While the RRF-EFG specific interactions are predominantly localized to the domains IV and V of EFG, the EFG-ribosome specific interactions that are crucial for ribosome recycling are not localized to a specific region of EFG but are found throughout the molecule. Our novel observations also emphasize the importance of using ribosomes from heterologous sources to understand the mechanism of this crucial process. (ii) Impact of rRNA methylations on ribosome recycling and fidelity of initiation in Escherichia coli Ribosomal RNA (rRNA) contains a number of modified nucleosides in functionally important regions including the intersubunit bridge regions; however, very little is known about the role of these rRNA modifications in ribosome function. As the activity of ribosome recycling factor (RRF) in separating the large and the small subunits of the ribosome involves disruption of the intersubunit bridges, we investigated the impact of rRNA methylations on ribosome recycling. The isolation of a folD122 mutant strain of E. coli with a deficiency in rRNA methylations, as well as the availability of E. coli strains deficient for various individual methyltransferases that modify specific rRNA residues, provided us with a genetic tool to assay the role of rRNA methylations in ribosome recycling. We observed that deficiency of rRNA methylations, especially at positions 1518 and 1519 of 16S rRNA near the interface with the 50S subunit and in the vicinity of the IF3 binding site, adversely affects the efficiency of RRF-mediated ribosome recycling. In addition, a compromise in the RRF activity was found to afford increased initiation with a mutant tRNAfMet wherein the three consecutive G-C base pairs (29GGG31:39CCC41), a highly conserved feature of the initiator tRNAs, were mutated to those found in the elongator tRNAMet (29UCA31:39ψGA41). This observation has allowed us to uncover a new role of RRF as a factor that contributes to fidelity of initiator tRNA selection on the ribosome. In addition, it was also found that IF3 and rRNA methylations, both of which are known to affect fidelity of initiation, exert their effects through distinct mechanisms, despite the proximity of a cluster of methylated rRNA residues to the IF3 binding site on the 30S subunit. (iii) Characterization of the role of EFG2, an EFG-like locus in Mycobacterium smegmatis Several bacteria, including various species of mycobacteria (with the exception of Mycobacterium leprae) contain a second EFG-like locus, denoted as fusA2, which shows considerable homology to fusA (encoding EFG). A comparison of the sequences of EFG and EFG2 from various bacteria reveals that EFG2 contains a GTPase domain and domains with significant homology to EFG domains IV and V, suggesting that it may function as an elongation factor. With the single exception of a recent study on Thermus thermophilus EFG2, this class of EFG-like protein factors has not been studied so far. Hence, it was of interest to characterize EFG2. In the current study, EFG2 from M. smegmatis was characterized both by in vitro biochemical assays as well as by in vivo experiments targeted to investigate the biological significance of EFG2 in mycobacteria. It was found that, unlike EFG, MsmEFG2 could not sustain either translocation or ribosome recycling in E. coli. Despite the fact that the purified MsmEFG2 could bind guanine nucleotides, it lacked the ribosome-dependent GTPase activity characteristic of EFG and other translation GTPases, suggesting that it was unlikely to function as an elongation factor. However, EFG2 was found to be expressed in stationary phase cultures of M. smegmatis. To understand the biological significance of EFG2, fusA2 was disrupted in M. smegmatis. The viability of the M. smegmatis mc2155 fusA2::kan derivative indicates that MsmfusA2 is a non-essential gene. While disruption of the fusA2 gene (encoding EFG2) in M. smegmatis does not appear to affect its growth and survival in log phase or stationary phase or under hypoxic conditions, preliminary experiments indicate that disruption of fusA2 confers a fitness disadvantage to M. smegmatis when competed against M. smegmatis mc2155 (with wild type fusA2 locus).
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