Non-coding RNAs (ncRNAs) account for a higher percent of the genome than coding mRNAs, and are implicated in human disease such as cancer, neurological, cardiac and many others. While the majority of ncRNAs involved in disease were originally attributed to a class of RNAs called micro RNAs (miRNAs) with a small size of only about 19 -24 base pairs, emerging research has now demonstrated a class of long non-coding RNAs (lncRNAs) that have a size of over 200 base pairs to be responsible for gene regulation and other functional roles and have also found to contribute to pathogenesis in humans. The increased size and structural complexity require novel tools to study their interactions beyond RNA interference. Synthetic antibodies are classic tools and therapeutics utilized to study and treat proteins involved in human disease. Likewise we hypothesize that structured RNAs can also take advantage of synthetic antibodies to probe their functions and be utilized as therapeutics. Currently, antibodies have been raised against microbial riboswitches and other structured RNAs of single-celled organisms, and only one human structured RNA to the best of our knowledge. However, no one has yet to create a synthetic antibody capable of behaving as a therapeutic against a structured RNA. We therefore sought to raise an antibody Fab against a structured RNA, human initiator tRNA, a model oncogenic non-coding RNA and demonstrate its efficacy in vitro. We then characterized the antibody and explored delivery options in cancer cells including the use of nanoparticle delivery systems. With the emerging transcriptome revealing new ncRNAs implicated in human disease, our research has begun to address a new therapeutic strategy, laying down the foundation for the future of structured RNA-targeted therapies.

The studies reported in this thesis address the aspects of initiator tRNA selection in Escherichia Coli. A summary of the relevant literature discussing the process of ptotein biosynthesis in general and initiator tRNA selection, in particular is presented in chapter 1. The next chapter (Chapter2) describes the ‘Materials and Methods’ used throughout the experimental work carried out in this thesis. It is followed by two chapters(Chapter 3 and Chapter 4) which describe the isolation and characterization of an E. coli mutant, to understand the mechanism of initiator tRNA selection. Chapter 5 comprises of some experimental work and future perspectives on the utility of the E.coli mutant. The last chapter (Chapter 6) summarizes the published work where I have contributed to besides the work described in Chapters 3 to 5. The summary of chapters 3-5 is as described below:- (i)Isolation and genetic mapping of extragenic suppressors of mutant initiator tRNA lacking the three consecutive G, C base pairs in the anticodon stem Initiator tRNA selection on the ribosomes is a result of several steps, some of which are unique to the prokaryotic world. Structure-function analyses of E.Coli tRNAfMet have revealed that the most important features of tRNAfMet, pertinent to its in vivo function as an initiator, are located in the acceptor stem and the anticodon arm regions. The three consecutive G-C base pairs in the anticodon stem of the tRNAfMet, conserved across all kingdoms of life, have been implicated in preferential binding to 30S ribosomal P-site. How the 3G-C base pairs are exploited by ribosomes in selecting the initiator tRNA, has been a long standing question. In the present work, a genetic screen was developed to isolate second site compensatory mutations of the mutant tRNAfMet, inactive in initiation because the 3G-C base pairs in it were changed to those found in the elongator tRNAMet(‘3G-C mutant’). Two extragenic suppressors were mapped to defined regions in the 12 min and 85 min locations in the E. Coli genome and three others were classified in these two broad groups. A super suppressor strain exhibiting synergistic suppression was generated. Further genetic mapping identified a G122D mutation in the folD gene encoding 5, 10 methylene tetrahydrofolate dehydrogenase/cyclohydrolase in one of the suppressor strains E. Coli A48. Complementation analysis using over expression of fold confirmed the results obtained by genetic mapping. (ii) Role of the intracellular S-adenosylmethionine flux in initiation with an initiator versus elongator tRNAs in Escherichia Coli How a defect in folD gene product (in E. Coli A48) leads to initiation with the ‘3G-C mutant’ initiator tRNA, has been addressed in this work. The FolD enzyme plays a key role in the one-carbon metabolism. The mutation in folD resulted in a lethal phenotype in minimal medium. The end-products of the pathway, 10 formyl-THF, methionine and S-adenosylmethionine(SAM) were analyzed for their possible role in initiation with the ‘3G-C mutant’ tRNAfMet, which revealed that lowering of the steady-state abundance of methionine and SAM had a direct role in initiation with the ‘3G-C mutant” tRNAfMet. Analysis of the 16S tRNA revealed that the methylations, as a result of reduced levels of SAM, were undetectable in the E.Coli A48. This prompted us to generate targeted mutations in the methyltransferase genes, which have highlighted the importance of methylations in initiator tRNA selection. Consistent with the growth retardation phenotype of methylase deficient strains at higher temperatures, the E. Coli A48 also displays temperature sensitivity. Further analysis of mycoplasma genomes, which do not follow the strong conservation of three G-C base pairs in the anicodon stem of initiator tRNA has uncovered an hitherto unknown evolutionary connection between methylations of 16S rRNA and initiator tRNA selection. We observed genetic interaction between infC(encoding IF3) and fold (encoding FolD). We also demonstrate initiation with tRNAfMet containing mutations in one, two or all the three G-C base pairs, as also with the elongator tRNA (tRNAGln). (iii) Utility of E. Coli A48 in investigation of biological processes: Some Preliminary studies and future perspectives. The availability of the E. Coli A48 strain is a valuable addition to the field of initiator tRNA selection and opens up further opportunities for its application. In this study, we have analyzed some of the properties of the E. Coli A48 strain viz. sensitivity to UV light and formylation independent initiation. E. Coli possess multiple copies of initiator tRNA, encoded by the metZVW operon and the metY gene. We reasoned that the abundance of cellular initiator tRNA might be a contributing factor in maintenance of specificity of initiation. Consistent with our prediction, we observed initiation with the ‘3G-C mutant’ tRNAfMet in E. Coli strains deficient in initiator tRNA genes. The various aspects of SAM limitation, biological functions of post-transcriptional modifications, incorporation of non-methionine amino acids in then-terminus of proteins and genetic approaches to system biology for the understanding of one-carbon metabolism are discussed.

Antibiotic resistance is a growing problem on a global scale. Increasing numbers of bacteria resistant toward one or multiple antibiotics could return us to the high mortality rates for infectious diseases of the pre-antibiotic era. The need for development of new classes of antibiotics is great as is increased understanding of the mechanisms underlying the development of antibiotic resistance. We have investigated the emergence of resistance to peptide deformylase inhibitors, a new class of antibiotics that target bacterial protein synthesis. The fitness of resistant mutants as well as their propensity to acquire secondary compensatory mutations was assessed in order to gain some insight into the potential clinical risk of resistance development. Most of this work was done in the bacterium Salmonella typhimurium, due to the availability of excellent genetic tools to study these phenomena. In addition, we have studied the bacterium Staphylococcus aureus as peptide deformylase inhibitors have been shown to have the greatest effect on Gram-positive organisms. In the course of this work we also examined the mechanistic aspects of translation initiation. Using a cell-free in vitro translation system we studied the effects of various components on translation initiation. These results have been combined with results obtained from resistant and compensated bacterial strains in vivo to gain new insights into the mechanisms of translation initiation.

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.

CSIR / Translation initiation is a rate limiting step during protein biosynthesis. Initiation occurs by formation of an initiation complex comprising 30S subunit of ribosome, mRNA, initiator tRNA, and initiation factors. The initiator tRNA has a specialized function of binding to ribosomal P site whereas all the other tRNAs are selected in the ribosomal A site. The presence of a highly conserved 3 consecutive G-C base pairs in the anticodon stem of the initiator tRNA has been shown to be responsible for its P-site targeting. The exact molecular mechanism involved in the P-site targeting of the initiator tRNA is still unclear and focus of our study. Using genetic methods, we obtained mutant E. coli strains where initiator tRNA mutants lacking the characteristic 3-GC base pairs can also initiate translation. One such mutant strain, A30, was selected for this study. Using standard molecular genetic tools, the mutation was mapped and identified to be a mutation in a transcription remodeling factor, RapA (A511V). RapA is a transcription recycling factor and it displaces S1 when it performs its transcription recycling activity. We found this mutation to cause an increase in the S1-depleted ribosomes leading to decreased fidelity of translation initiation as the mutant RapA inefficiently displaces S1 from RNA polymerase complex. The mutation in the RapA was also found to cause changes in the transcriptome which leads to downregulation of major genes important for methionine and purine metabolism. Using mass spectrometric analysis, we identified deficiencies of methionine and adenine in the strain carrying mutant RapA. Our lab had previously reported that methionine and S-adenosyl methionine deficiency cause deficiency of methylations in ribosome which in turn decreases the fidelity of protein synthesis initiation. We used strains deleted for two newly identified methyltransferases, namely RlmH and RlmI, for our study and these strains also showed decreased fidelity of initiation. RlmH and RlmI methylate 1915 and 1962 positions of 23S rRNA respectively. We found that deletion of these methyltransferases also caused defects in ribosome biogenesis and compromised activity of ribosome recycling factor. We constructed phylogenetic trees of the initiator tRNA from 158 species which distinctly assembled into three domains of life. We also constructed trees using the minihelix or the whole sequence of species specific tRNAs, and iterated our analysis on 50 eubacterial species. We identified tRNAPro, tRNAGlu, or tRNAThr (but surprisingly not elongator tRNAMet) as probable ancestors of tRNAi. We then determined the factors imposing selection of methionine as the initiating amino acid. Overall frequency of occurrence of methionine, whose metabolic cost of synthesis is the highest among all amino acids, remains almost unchanged across the three domains of life. Our results indicate that methionine selection, as the initiating amino acid was possibly a consequence of the evolution of one-carbon metabolism, which plays an important role in regulating translation initiation. In conclusion, the current study reveals the importance of methylations in ribosome biogenesis and fidelity of translation initiation. It also strongly suggests a co-evolution of the metabolism and translation apparatus giving adaptive advantage to the cells where presence of methionine in the environment can be a signal to initiate translation with methionine initiator tRNA.