Dissertations / Theses on the topic 'Initiator tRNAs'

Initiation of mRNA translation in prokaryotes requires the small ribosomal subunit (30S), three initiation factors, IF1, IF2 and IF3, initiator tRNA (fMet-tRNAfMet) and the large ribosomal subunit (50S). During initiation, the 30S ribosomal subunit interacts with the three factors and binds in a presumably random manner fMet-tRNAfMet and mRNA. This initially formed 30S pre-initiation complex is a kinetic intermediate of the stable 30S initiation complex which is formed upon base pairing of the anticodon of fMet-tRNAfMet and the intiation codon of the mRNA in the P-site. In a subsequent step the 50S subunit binds to the 30S initiation complex giving rise to a 70S initiation complex. During this step, a molecule of GTP bound to IF2 is hydrolyzed, the initiation factors are released from the ribosome and fMet-tRNAfMet is stabilized in the P site ready to enter in the elongation phase of translation. In turn, each of these major steps involves numerous elemental steps, whose sequence and timing are largely unknown. How the fMettRNAfMet is bound to the ribosome during the assembly of the initiation complex is one of the many questions still remaining open. According to the classical model, IF2 acts as a carrier of fMet-tRNAfMet to the ribosome. An alternative model predicts that the IF2, prebound to the 30S subunit awaits the arrival of fMet-tRNAfMet with which it interacts only at the ribosomal surface. This work presents results of a kinetic analysis of binding to the 30S subunit of various ribosomal ligands (Initiation Factors, fMet-tRNAfMet, mRNA), performed with fast kinetic techniques (rapid filter binding, fluorescence stopped flow). The data obtained strongly support the model in which IF2 is not a fMettRNAfMet carrier. The elemental rate constants determined here give us a more detailed description of the process of 30S initiation complex assembly, of the interplay of the various ribosomal ligands and of the functional significance of different structural modules of IF2.

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.

Translation initiation factor 1 (IF1) has been shown to be an RNA chaperone. In order to find functional interactions that IF1 may have with rRNA, we have isolated second-site suppressors of a cold-sensitive IF1 mutant. Joining of the ribosomal subunit seems to be affected in the IF1 mutant strain and the suppressive effect is a consequence of decreasing the available pool of mature 50S subunits. The results serve as additional evidence that IF1 is an RNA chaperone and that final maturation of the ribosome takes place during translation initiation. In this study we have also investigated the effect of a cold-sensitive mutant IF1 or kasugamycin addition on gene expression using a 2D gel electrophoresis technique. The effect is much more dramatic when cells are treated with kasugamycin compared to mutant IF1. The ybgF gene is uniquely sensitive to the IF1 mutation as well as the addition of kasugamycin. This effect on the native gene could be connected with some property of the TIR sequence of ybgF and supports the notion that kasugamycin addition and the IF1 cold-sensitive mutation have a similar TIR-specific effect on mRNA translation. Finally we have isolated a suppressor of a temperature-sensitive mutation in ribosomal release factor 1 (RF1) to shed more light on the translation termination process. The suppressor mutation is linked to an IS10 insertion into the cysB gene and results in a Cys- phenotype. Our results suggest that suppression of the thermosensitive growth is a consequence of the mnm5s2U hypomodification of certain tRNA species. The ability of mnm5s2U hypomodified tRNA to induce frameshifting may be responsible for the suppression mechanism and it supports the hypothesis that modified nucleosides in the anticodon of tRNA act in part to prevent frameshifting by the ribosome.
At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.

All eukaryotic mRNAs process a cap structure (m7G(5')ppp(5')N) at the S' end of their message and most have an A as the first nucleotide after the cap. However, 30% of messages within eukaryotic cells have a C (m7G(t')ppp(5')C) as the first nucleotide followed by a short polypyrimidine tract. These mRNAs are termed TOP (Tenninal Oligopyrimidine tract) messages and are co-ordinately regulated by mitogenic, growth and nutritional stimuli. This work describes the construction of a reporter vector that encodes mRNA containing the TOP motif, and its use in a series of systematic experiments to further investigate the translational regulation of TOP messages. Given that TOP containing mRNAs are known to encode proteins involved in the translational machinery, these findings have important implications with regard to translational control and translation related disease. In this study, reporter vectors have been used to investigate the role of the mTOR and PI3K signalling pathways, which have previously been implicated in the translational regulation of TOP containing mRNAs. The data obtained suggests that the mTOR signalling pathway may be involved in the regulation of TOP containing mRNAs. The canonical initiation and frans-acting factor requirements of TOP mRNAs were also investigated using a combination of protein over-expression and affinity purification of TOP-containing-mRNA:protein complexes. The data obtained raises the possibility that eIF4E may not be required in the initiation of TOP containing mRNA translation. The candidate trans-acting factors that were identified include La, ILF2 and EBPl, the latter of which has previously been shown to associate with mature ribosomes in the cytoplasm. Finally, affinity purification of TOP-containing-mRNA:microRNA complexes was carried out. Candidate microRNAs which may be involved in the regulation of TOP containing mRNAs were identified. The data obtained was consistent with a previous study, which suggested that microRNA-IOa may bind to the S'UTR of TOP containing mRNAs.

Mass production of translationally optimized bacteriophages (hereafter referred to as phages) is the need of the hour in the application of phages to therapy. Understanding translational efficiency of phages is the major preliminary step for mass producing efficient phages. The objective of this thesis is to understand factors affecting translational efficiency of phages. In chapter two, we hypothesized that weak translation initiation efficiency is responsible for weak codon concordance of Escherichia coli lambdoid phages with that of their hosts. We measured the strength of translation initiation using two indices namely minimum folding energy (MFE) and proportion of Shine-Dalgarno sequence (PSD). Empirical results substantiate our hypothesis suggesting lack of strong selection for improving codon adaptation in these phages is due to their weak translation initiation. In chapter three, we measured codon usage concordance between GC-rich and GC-poor Aeromonas phages with their GC-rich host Aeromonas salmonicida. We found low codon usage concordance in the GC-poor Aeromonas phages. We were interested in testing for the role of tRNAs in the GC-poor phages. We observed that the GC-poor phages carry tRNAs for codons that are overused by the phages and underused by the host. These findings suggest that the GC-poor Aeromonas phages carry their own tRNAs for compensating for the compositional difference between their genomes and that of their host. Previously several studies have reported observed avoidance of stable secondary structures in start site of mRNA in a wide range of species. We probed the genomes of 422 phage species and measured their secondary structure stability using MFE. We observed strong patterns of secondary structure avoidance (less negative MFE values) in the translation initiation region (TIR) and translation termination region (TTR) of all analyzed phages. These findings imply selection is operating at these translationally important sites to control stable secondary structures in order to maintain efficient translation.

La chaîne de Taïwan est un exemple unique où il est possible d’étudier une transition subduction/collision active. L’obliquité de la convergence permet en effet d’observer du Sud vers le Nord les stades initiaux de l’accrétion continentale en mer jusqu’au stade mature de la collision. Je montre que la Chaîne Centrale au Sud de Taiwan, située la zone de transition, exhume des assemblages pétrographiques de grades croissants vers le Nord, dans un couloir décrochant senestre qui résulte du partitionnement de la convergence. Dans ce couloir l’extension sub-parallèle à la chaîne accommode une partie de l’exhumation. A proximité de la zone de suture principale, des assemblages de haute pression (12-15 kbars et 380-420 °C) associés à des blocs exotiques de serpentinites se sont mis en place dans un encaissant d’âge Miocène HT-BP associé à une intense déformation cisaillante senestre. Les analyses pétrologiques et géochimiques des mélanges ophiolitiques de Kenting et Lichi montrent que ces mélanges proviennent de deux sources situées à l’origine au pied de la marge continentale et dans la croûte océanique. Enfin, je propose, un nouveau modèle tectonique 3D du sud de Taïwan qui tient compte de l’architecture de la marge continentale, et qui met en avant la formation de la chaîne de Taiwan en contexte décrochant
The Taiwan collision is the unique example where to examine ongoing tectonic processes at the subduction/collision transition. The oblique convergence setting allows observations of first continental accretion offshore to the mature stage of collision onshore. I show that the southern Central Range, located at the transition, exhumed petrographic assemblages with peak metamorphism increasing northwards, in a corridor characterized by left-lateral transcurrent deformation. Extension sub-parallel to the belt is also understood as a major ingredient of rock exhumation. Near the suture zone, HP rocks (12-15 kbars and 380-420 °C) associated with isolated serpentinite bodies were emplaced in association with intense ductile strain localization in the strike-slip corridor. Petrographc and geochemical analyses of the Kenting and Lichi ophiolitic mélanges reveal that these mélanges originated from the distal rifted margin and the oceanic crust of the South China Sea. I propose a 3D geodynamic model of the evolution of the Taiwan orogen in which the rifted margin architecture is taken into account and the strike-slip kinematic component of the convergence is the main driver of mountain building

La glycyl-ARNt synthétase humaine (GRS) est une enzyme clé dans la traduction des protéines dans le cytosol et la mitochondrie. Chez l’Homme, des mutations de la GRS conduisent à la neuropathie périphérique Charcot-Marie-Tooth (CMT). Bien que l’activité de la GRS soit ubiquitaire, les mutations associées à la CMT n’affectent que les nerfs périphériques, suggérant un rôle supplémentaire de la GRS dans les neurones. Pour comprendre ce rôle, nous avons d’abord élucidé le mécanisme particulièrement complexe qui contrôle l’expression de la GRS mitochondriale et cytosolique à partir du même gène. Nous avons identifié deux ARNm : un codant pour les deux enzymes ; et un autre plus long qui contient une IRES fonctionnelle et un uORF. Cet ARNm complexe, ne génère que la GRS cytosolique et montre que son expression et localisation sont étroitement contrôlées. De plus, nous avons montré une distribution particulière de la GRS dans des neurones, qui est un premier indice sur un rôle non canonique
Human Glycyl-tRNA synthetase (GRS) is a housekeeping enzyme with a key role in protein synthesis, both in the cytosol and the mitochondria. In human, mutations in GRS cause the Charcot-Marie-Tooth (CMT) peripheral neuropathy. Though GRS activity is required in all cells, the CMT-associated mutations affect only the peripheral nervous system, suggesting an additional non canonical role.To understand how GRS is involved in CMT pathology, we first elucidated the original post-transcriptional regulatory mechanism that controls the expression of both the mitochondrial and the cytosolic GRS from a single gene. We identified two mRNA isoforms: one coding for both enzymes; and a longer one containing a functional IRES and an uORF encoding only the cytosolic GRS, evidence that expression and localization of human GRS are tightly controlled. Furthermore, we found a particular Ca2+ dependant distribution of GRS in neurons, giving us a first clue about a potential non-canonical role in neurons

L'hydrogenase de rhodobacter capsulatus est une enzyme a nickel et a fer qui, en oxydant l'hydrogene, permet a la bacterie de croitre en autotrophie. La synthese de l'hydrogenase est soumise a plusieurs niveaux de regulation. En absence de carbone organique l'enzyme est toujours synthetisee. En heterotrophie, l'expression des genes de structure de l'hydrogenase, les genes hupslc, a lieu seulement en presence d'h 2. La proteine hupr est un facteur de transcription essentiel a l'expression de l'operon hupslc. Des fusions traductionnelles phups : : lacz ainsi qu'une etude in vitro de la fixation de la proteine hupr purifiee au promoteur de hups ont montre que le regulateur se fixe sur une seule sequence palindromique, ttg-n 5-caa, en position -162/-152 nt du site d'initiation de la transcription. Hupr est un regulateur de reponse de la sous-famille ntrc des systemes a deux composants et il interagit par transfert de phosphate avec la proteine histidine kinase hupt. Des experiences realisees avec des proteines huprd54 mutees confirment que l'aspartate 54 est bien le site de phosphorylation de la proteine. Nous avons mis en evidence les deux particularites du systeme a deux composants hupt/hupr. D'une part, le regulateur est actif a l'etat non phosphoryle et la phosphorylation l'inactive. En effet, les proteines mutees huprd54 ou huprnter restaurent le phenotype sauvage d'une souche hupr , en conditions d'activation de l'hydrogenase. D'autre part, le regulateur hupr active une arn polymerase a 7 0 et non a 5 4 comme c'est le cas generalement pour les regulateurs de reponse des systemes a deux composants. La regulation de la synthese de l'hydrogenase en reponse a l'h 2 fait aussi intervenir l'hydrogenase regulatrice hupuv. Afin de reconstituer in vitro cette cascade de regulation, nous avons surproduit et purifie les proteines hupuv sur colonne d'affinite, mais elles etaient inactives. Le systeme de regulation global regb/rega est implique dans la repression de la synthese de l'hydrogenase. L'action du systeme regb/rega se superpose a celle du systeme specifique hupt/hupr. L'objectif est donc maintenant de comprendre comment se font les interactions entre les diverses proteines fixees au promoteur phups (hupr, ihf, rega et la polymerase a 7 0). Il faudra notamment preciser comment hupr active la polymerase a facteur 7 0 et comment rega reprime la synthese de l'hydrogenase.

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.

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.

The studies reported in this thesis describe the work done in the area of translation initiation where a previously unknown role of multiple copies of initiator tRNA in E. coli has been reported. Also the role of SsrA resume codon in resumption of translation, until not clearly known has been reported here. Chapter -1 discusses the relevant literature in understanding translation and initiator tRNA selection on the ribosome during initiation. It also discusses the literature pertaining to the aspect of release of stalled ribosomal complexes by SsrA. This is followed by the next chapter (chapter- 2) which discusses the materials and methods used throughout the study. Chapter- 3 describes the studies leading to the role of multiple copies of initiator tRNA in E. coli in governing the fidelity of initiator tRNA selection on the P site of the ribosome. This is followed by Chapter-4 which describes the role of the resume codon of the SsrA in governing the efficiency of trans-translation in releasing the stalled ribosomal complexes. The summaries of the chapters 3 and chapter 4 are briefly described below. i) Role of conserved 3GC base pairs of initiator tRNA in the initiator-elongator tRNA discrimination. Translation initiation is the first step in the very important and highly conserved biological process of protein biosynthesis. The process involves many steps, a wide array of protein factors at each specialized step and a large ribonucleoprotein particle; the ribosome to decode the information of the mRNA template into biologically active proteins. The process of initiation is still unclear largely due to fewer reports of available structural data. One of the very interesting questions that people have been trying to address is how the initiator tRNA is selected on the P- site of the ribosome and what is the importance of the conserved three GC base pairs in the anticodon stem of the initiator tRNA. Here in this study, I have studied this question by using the classical genetic technique of generating and characterizing the mutant initiator tRNA defective at the step of initiation. I have identified and analyzed the suppressors which are capable of rescuing this defect in initiation. The study involves two such E. coli suppressor strains (named D4 and D27). These suppressors can initiate translation from a reporter CAT mRNA with amber codon, independent of the presence of the three consecutive GC base pairs in the anticodon stem of initiator tRNAs. Mapping of the mutations revealed that the mutants are defective in expression of the tRNA1fMet (metZVW) gene locus which encodes the initiator tRNA. Both the suppressors (D4 and D27) also allow initiation with elongator tRNA species in E. coli. Taken together, the results show that E. coli when deficient in the initiator tRNA concentration can lead to initiation with elongator tRNA species. ii) The Role of SsrA/tmRNA in ribosome recycling and rescue. Occasionally during the process of translation, the ribosomes stall on the mRNA before the polypeptide synthesis is complete. This situation is detrimental to the organism because of the sequestration of the tRNAs as ‘peptidyl tRNAs’ and the ribosomes. In E. coli one of the pathways to rescue stalled ribosomes involves disassembly of these stalled complexes to release peptidyl tRNAs which are then recycled by peptidyl tRNA hydrolase (Pth), an essiential enzyme in E. coli. The other pathway which is not essential in E. coli but is conserved in all prokaryotes involves SsrA or tmRNA (transfer messenger RNA). The tmRNA is charged with alanine and recognizes the stalled ribosomal complexes and acts as tRNA to bind the A-site. It also functions as mRNA by adding a undecapeptide (which is actually a tag for degradation by cellular proteases) to the existing polypeptide and there is normal resumption of the translation. In most sequences of SsrA ORF, the first codon of the ORF, called as resume codon, is conserved. I wanted to understand the importance of the conservation of the resume codon. Towards this end I randomly mutated the resume codon and studied the effect of the altered resume codon in the rescue of stalled ribosomal complexes. The effect of over-expression of these mutants was investigated in the rescue of the Pthts defect since it is known that the overexpression of SsrA rescues the temperature sensitive phenotype of the Pthts strain and so causes less accumulation of peptidyl–tRNA in E. coli .The effect for these mutants has also been studied by the growth of hybrid λimmP22 phages. I also used AGA minigene system to study the effect of various mutants which has been shown to sequester tRNAArg (UCU) in the ribosomal P-site, translation of this minigene causes toxicity to E. coli. I have tried to study the effect of the SsrA mutants in rescue of toxicity caused by the minigene. Overall, the observations indicate that the conservation of the resume codon is important in E. coli and having mutated resume codon probably leads to deficient trans-translation during one or the other growth conditions.

碩士
國立中興大學
分子生物研究所
84
Abstract The effect of 5' flanking DNA sequence on the transcription activity and tRNA production of the monocistronic spinach chloroplast trnS gene was evaluated. Deletion of the 5' DNA flanking sequence to the position of 47 nucleotides from the trnS coding region does not affect the production of mature tRNA. Although the transcription remains active for the template contains no 5' flanking sequence of trnS gene, the deletion of the 5' DNA flanking sequence beyond 47 nucleotides from the trnS coding region significantly reduce the production of mature tRNA. This observation is not consistent to the previous report that the transcription of trnS gene required no 5' upstream DNA sequences. The chloroplast transcriptionally active enzyme extract contains both transcription and tRNA processing activities. The primary transcripts produced by RNA polymerase are immediately processed to mature tRNA by the tRNA processing enzymes in the same extract. No primary transcript can be obtained under this reaction condition. An approach by deleting part of the tRNA 3' end coding region that changing the tRNA secondary structure and hence abolishes the processing activity was developed to obtain the primary transcript of trnS gene. The transcript produced by this approach was confirmed by in vitro capping assay and used as template for determination of its transcription start site. The transcription start site for wild-type trnS gene is located at the 11 nucleotides 5' end to the coding region, whereas, the transcription start sites were varied for the primary transcripts prepared from various 5' deletion mutants. An in vitro tRNA processing system using spinach chloroplast enzyme extract has been previously established in our laboratory. In this study, the serine tRNA precursors prepared from T7 RNA polymerase as well as from spinach chloroplast RNA polymerase were incubated with chloroplast enzyme extract for tRNA processing activity assay. The results indicated that both the 5' and 3' ends of serine tRNA were generated by endonucleases and the 5' end cleavage was demonstrated to precede the 3' end cleavage.

碩士
慈濟大學
醫學研究所
85
Two initiator species of formylmethionine tRNA, tRNAfMet, are present in Escherichia coli. These differ in a single nucleotide at position 46. The major species, tRNA1fMet, has m7G46, whereas tRNA2fMet, the minor species, has A. This single, base renders two initiator tRNAs with different conformation. Previous studies show that tRNA1fMet is encoded by three copies of met Z gene and tRNA2fMet is encoded by a single copy of met Y gene. The physiological significance to have these two initiator species in E. coli is not established. Interestingly, it was reported that tRNA2Met gene was mutated to tRNA1fMet gene in one of the E. coli B strains, B105. This conclusion was based on the direct sequence comparison on the met Y gene between E. coli K-12 (JM103) and E. coli B (B105). However, the results on the genomic organization and sequence in the met Z genes have been controversial and not established. In this study, the tRNA1fMet met Z was cloned from different E. coli species. Our PCR and sequencing data show that there are three identical copies of tRNA1fMet gene in both E. coli K12 (JM103) and E. coli B (B105). We have thus ruled out the possibility that a tRNA species which co-migrates with the wild type tRNA2fMet detected on the Northern blot under acidic conditions is due to the mutation(s) on one of the tRNA1fMet genes on the met Z locus. Our data also suggest that the tRNA2fMet-like species is most likely a undermodified tRNA1fMet species which is caused by the limited capacity of the base modifying enzyme(s) found in E. coli B105.

Studies reported in this thesis address the question of how pre-termination ribosomal complexes stalled during translation of mRNA are recycled. The process of recycling of the stalled ribosomes involves many translational factors. During the course of my studies, I have uncovered new roles of SsrA (tmRNA), IF3 and ribosome recycling factor (RRF) in recycling stalled ribosomes. These findings are summarized as follows: (i) A physiological connection between tmRNA and peptidyl-tRNA hydrolase functions in Escherichia coli The bacterial ssrA gene codes for a dual function RNA, tmRNA, which possesses tRNA-like and mRNA-like regions. The tmRNA appends an oligopeptide tag to the polypeptide on the P-site tRNA by a trans-translation process that rescues ribosomes stalled on mRNAs and targets the aberrant protein for degradation. In cells, processing of the stalled ribosomes is also pioneered by drop-off of peptidyl-tRNAs. The ester bond linking the peptide to tRNA is hydrolyzed by peptidyl-tRNA hydrolase (Pth), an essential enzyme, which releases the tRNA and the aberrant peptide. As the trans-translation mechanism utilizes the peptidyl-transferase activity of the stalled ribosomes to free the tRNA (as opposed to peptidyl-tRNA drop-off), the need for Pth to recycle such tRNAs is bypassed. Thus, we hypothesized that tmRNA may rescue a defect in Pth. The findings of the experiments detailed in this thesis show that SsrA rescues a defect in Pth by reducing the peptidyl-tRNA load on Pth. (ii) Evidence for a role of initiation factor 3 in recycling ribosomal complexes stalled on mRNAs in Escherichia coli. Specific interactions between ribosome recycling factor (RRF) and EF-G mediate disassembly of post-termination ribosomal complexes for new rounds of initiation. The interactions between RRF and EF-G are also important in peptidyl-tRNA release from pre-termination complexes. Unlike the post-termination complexes (harboring tRNA), the pre-termination complexes (harboring peptidyl-tRNA) are not recycled by RRF and EF-G in vitro, suggesting participation of additional factor(s) in the process. Using a combination of biochemical and genetic approaches, we show that, 1. Inclusion of IF3 with RRF and EF-G results in recycling of the pre-termination complexes; 2. IF3 overexpression in Escherichia coli LJ14 rescues its temperature sensitive phenotype for RRF; (3) Transduction of infC135 (encoding functionally compromised IF3) in E. coli LJ14 generates a ‘synthetic severe’ phenotype; (4) The infC135 and frr1 (a promoter down RRF gene) alleles synergistically rescue a temperature sensitive mutation in peptidyl-tRNA hydrolase in E. coli; and (5) IF3 facilitates ribosome recycling by Thermus thermophilus RRF and E. coli EFG in vivo and in vitro. These lines of evidence clearly demonstrate the physiological importance of IF3 in the overall mechanism of ribosome recycling in E. coli. (iii) The role of RRF in dissociating of pre-termination ribosomal complexes stalled during elongation Translating ribosomes often stall during the repetitive steps of elongation for various reasons. The stalled ribosomes are rescued by the process of trans-translation involving tmRNA (SsrA) or by a factor mediated dissociation of the stalled ribosome into its subunits leading to the drop-off of the peptidyl-tRNA. The mechanistic details of how the factor mediated dissociation is carried out, is not well studied. Studies described in the above section have highlighted the role of RRF in dissociating stalled pre-termination complexes. However, the in vivo studies in this area have been limited for lack of defined pre-termination complexes. Two in vivo systems based on translation of AGA minigene and the ung gene (EcoUngstopless) transcripts were designed. Evidence is presented to show that translation of both of these transcripts is toxic to E. coli because of the accumulation of the transcript specific stalled pre-termination complexes. Availability of these model systems has allowed us to address the role of RRF in dissociating stalled ribosomes. We show that RRF rescues stalled ribosomes on these constructs and its overexpression can rescue the toxicity. The physiological importance of this observation is highlighted by the rescue of AGA minigene inhibitory effect on λimmP22 hybrid phage growth upon RRF overexpression.

Hepatitis C virus (HCV) is a worldwide spread pathogen infecting up to 3 % of the human population. Nowadays, research of new drugs against this virus is focused on the individual steps in its life cycle, including the translation initiation. In the case of HCV translation initiation is dependent on the internal ribosome entry site (IRES). Besides of components of the translational machinery also other components of the cell, so called IRES trans-acting factors (ITAF), contribute to its proper progress. This work continues in previous research of our laboratory focused on searching for new ITAF. In order to search for potential ITAF increasing HCV IRES activity new recombinant plasmid vectors and reference strains were prepared and selection conditions of the selection system were optimized. The differences in the growth characteristics of the reference strains were analyzed and quantified under selective and non-selective conditions. A set of pilot high efficiency transformations of the yeast strain pJ69-4A carrying bicistronic construct with HCV IRES were conducted using human expression cDNA library in order to optimize the efficiency of transformation and selection conditions and to attempt to identify new ITAF. Several dozens of randomly selected clones from these transformations obtained under...