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.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Inorganic Phosphate (Pi) is one of the key nutrients required by all living organisms on earth. This nutrient is of vital importance to higher plants but it is not readily available for uptake from the soil, implying constant stress on plants. During photosynthetic dark and light reactions, phosphate is a prerequisite for all reactions to occur and to ensure plant survival. This statement implies that a careful homeostatic control of this nutrient is necessary in order to maintain a balanced carbon flow in all sub-cellular plant compartments. Phosphate limitation is a threat to plant survival and one way of addressing this nutritional hurdle is by feeding plants with fertilizer. This method of crop development and general plant maintenance by humans has a devastating effect on the environment, as phosphate causes eutrophication and various other consequences which are detrimental to animal life. Plants, however, are naturally equipped with Pi transporters which are activated conditionally depending on the external Pi availability. These transporters are present in most sub-cellular compartments and some of them have been identified and characterised, while others remain to be a prediction. If these transporters are characterised accordingly it might eventually mean that the use of fertilizers may no longer be necessary. In order to contribute to successful Pi-efficient crop development, a clearer understanding of P-dynamics in the soil and its recycling ability inside the plant itself is necessary. During this study it was attempted to characterise a putative high affinity Pi transporter, PHT1;5, from Arabidopsis thaliana via a Escherichia coli and yeast heterologous expression system and its Km value predicted in order to verify/hypothesise whether it is a high or low affinity transporter. This transporter is expressed in leaves and could be a promising tool for future carbon partitioning studies during phosphate limitation. / AFRIKAANSE OPSOMMING: Anorganiese fosfaat (Pi) word beskou as een van die belangrikste nutriente benodig vir alle lewe op aarde. Dit vervul ‘n hoof rol in talle noodsaaklike prosesse in hoër plante en is veral ‘n voorvereiste vir fotosintetiese reaksies om plaas te vind. In ‘n plant se natuurlike omgewing is anorganiese fosfaat nie geredelik bekskikbaar in grond nie en dus word daar vermoed dat plante onder konstante fosfaat stres gevind word. Omdat fosfaat so ‘n belangrike rol speel tydens fotosintese is dit noodsaaklik dat daar ‘n balans op sellulêre vlak gehandhaaf word, veral wat die verspreiding van koolhidrate tussen die verskillende kompartemente van die sel betref. Plante se oorlewing word bedreig deur ‘n tekort aan fosfaat in die omgewing en die enigste onmiddelike oplossing daarvoor is deur die toediening van bemestingstowwe. Hierdie metode van landery ontwikkeling en algemene instandhouding van plante deur die mensdom het ’n baie negatiewe effek op die omgewing. ‘n Oormaat fosfaat lei tot eutrifikasie en het verkeie ander negatiewe nagevolge wat dodelik is vir die dierelewe. Plante beskik ook oor natuurlike interne fosfaat transporters om hierdie tekort te oorkom. Hierdie transporters word op grond van eksterne fosfaat beskikbaarheid ge-aktiveer of ge-deaktifeer. Die transporters is teenwoordig in meeste sub-sellulêre kompartemente en sommige is al ge-identifiseer en gekarakteriseer, terwyl ander slegs ‘n voorspelling bly. Gedurende hierdie studie was ‘n poging aangewend om ‘n anorganiese fosfaat transporter van Arabidopsis thaliana, PHT1;5, te karakteriseer met behulp van mikro-organismes soos Escherichia coli en gis. Die poging het ingesluit om ‘n Km waarde vir hierdie transporter te voorspel en sodoende ‘n hipotese daar te stel van of dit hoë of lae affiniteit het vir fosfaat. Die transporter word groot en deels aangetref in blare en kan dus dien as ‘n belowende apparaat vir toekomstige koolhidraat uitruiling studies gedurende fosfaat tekort.