How Much Initiator tRNA Does Escherichia Coli Need?

Abstract

The work discussed in this thesis deals with the significance of initiator tRNA gene copy number in Escherichia coli. A summary of the relevant literature discussing the process of protein synthesis, initiator tRNA selection and gene redundancy is presented in Chapter 1. Chapter 2 describes the ‘Materials and Methods’ used in the experimental work carried out in this thesis. The next three chapters address the significance of initiator tRNA gene copy number in E. coli at three levels; at the level of the molecule (Chapter 3), at the level of the cell (Chapter 4) and at the level of the population (Chapter 5). At the end of the thesis are appended three publications, which include two papers where I have contributed to work not discussed in this thesis and one review article. A brief summary of chapters 3 to 5 is provided below: (i) Chapter 3: Can E. coli remain viable without the 3 G-C base pairs in initiator tRNA? Initiator tRNAs are distinguished from elongator tRNAs by several features key among which are the three consecutive and near universally conserved G-C base pairs found in the anticodon stem of initiator tRNAs. These bases have long been believed to be essential for the functioning of a living cell, both from in vitro and in vivo analysis. In this study, using targeted mutagenesis and an in vivo genetics based approach, we have shown that the 3 G-C base pairs can be dispensed with in E. coli, and the cell can be sustained on unconventional initiator tRNAs lacking the intact 3 G-C base pairs. Our study uncovered the importance of considering the relative amounts of molecules in a living cell, and their role in maintaining the fidelity of protein synthesis. (ii) Chapter 4: Can elongator tRNAs initiate protein synthesis? There are two types of tRNAs; initiator tRNA, of which there is one representative in the cell, and elongator tRNAs of which there are several representatives. In this study, we have uncovered initiation of protein synthesis by elongator tRNAs by depleting the initiator tRNA content in the cell. This raises the possibility that competition between initiator and elongator tRNAs at the P site of the ribosome occurs routinely in the living cell, and provides a basis for initiation at several 'start' sites in the genome that may not be currently annotated as such. We speculate that such a phenomenon could be exploited by the cell to generate phenotypic diversity without compromising genomic integrity. (iii) Chapter 5: How many initiator tRNA genes does E. coli need? E. coli has four genes that encode initiator tRNA, these are the metZWV genes that occur at 63.5 min in the genome, and the metY gene that occurs at 71.5 min in the genome. Earlier studies indicated that the absence of metY had no apparent impact on cell growth. In view of the importance of initiator tRNA gene copy number in maintaining the rate and fidelity of protein synthesis, we examined the fitness of strains carrying different numbers of initiator tRNA genes by competing them against each other in both rich and limited nutrient environments. Our results indicate a link between caloric restriction and protein synthesis mediated by the initiator tRNA gene copy number.