Transfer RNAs (tRNA) function as adaptor molecules in the translation of mRNA into protein. These adaptor molecules require modifications of a subset of their nucleosides for optimal function. The most frequently modified nucleoside in tRNA is position 34 (wobble position), and especially uridines present at this position. Modified nucleosides at the wobble position are important in the decoding process of mRNA, i.e., restriction or improvement of codon-anticodon interactions. This thesis addresses the functional aspects of the wobble uridine modifications. The Saccharomyces cerevisiae Elongator complex consisting of the six Elp1-Elp6 proteins has been proposed to participate in three distinct cellular processes; elongation of RNA polymerase II transcription, regulation of polarized exocytosis, and formation of modified wobble nucleosides in tRNA. In Paper I, we show that the phenotypes of Elongator deficient cells linking the complex to transcription and exocytosis are counteracted by increased level of and . These tRNAs requires the Elongator complex for formation of the 5-methoxycarbonylmethyl (mcmlnGUUGsmcm25tRNALysUUUsmcm25tRNA5) group of their modified wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). Our results therefore indicate that the relevant function of the Elongator complex is in formation of modified nucleosides in tRNAs and the defects observed in exocytosis and transcription are indirectly caused by inefficient translation of mRNAs encoding gene products important for these processes. The lack of defined mutants in eukaryotes has led to limited understanding about the role of the wobble uridine modifications in this domain of life. In Paper II, we utilized recently characterized mutants lacking the 2-thio (s2) or 5-carbamoylmethyl (ncm5) and mcm5 groups to address the in vivo function of eukaryotic wobble uridine modifications. We show that ncm5 and mcm5 side-chains promote reading of G-ending codons, and that presence of a mcm5 and an s2 group cooperatively improves reading of both A- and G-ending codons. Previous studies revealed that a S. cerevisiae strain deleted for any of the six Elongator subunit genes shows resistance towards a toxin (zymocin) secreted by the dairy yeast Kluyveromyces lactis. In Paper III, we show that the cytotoxic γ subunit of zymocin is a tRNA endonuclease that target the anticodon of mcm5s2U34 containing tRNAs and that the wobble mcm5 modification is required for efficient cleavage. This explains the γ-toxin resistant phenotype of Elongator mutants which are defective in the synthesis of the mcm5 group.

Transfer RNA (tRNA) is the adaptor molecule responsible for bringing the correct amino acid to the ribosome during protein synthesis. tRNA contains a number of modified nucleosides, which are derivatives of the four normal nucleosides. A great variety of modifications are found in the anticodon loop, especially at the first (wobble) position of the anticodon. According to Crick’s wobble hypothesis, a uridine at the wobble position of tRNA recognize codons ending with A and G. Uridine-5-oxyacetic acid (cmo5U34), found at the wobble position of six species of tRNA in Salmonella enterica, have been predicted to expand the codon recognition of uridine to include U-ending, but not C-ending codons. To study the function of cmo5U34 we have identified two genes, cmoA and cmoB, which are required for the synthesis of cmo5U34 in tRNA. We have shown that the proline, alanine and valine tRNAs containing cmo5U34 are capable of reading codons ending with any of the four nucleotides, while the threonine tRNA is not, and the importance of having cmo5U is different for the different tRNAs. In addition, we found that cmo5U is important for efficient reading of G-ending codons, which is surprising considering the wobble hypothesis, which states that uridine should read G-ending codons. The dominant +1 frameshift suppressor sufY suppresses the hisC3737 +1 frameshift mutation. We have demonstrated that sufY induces frameshifting at CCC-CAA (Pro-Gln), when tRNAPro[cmo5UGG] occupies the P-site. sufY mutants accumulate novel modified nucleosides at the wobble position of tRNAs that should normally have (c)mnm5s2U34. The presence of an extra sidechain (C10H17) on the wobble nucleoside of tRNAGln[(c)mnm5s2U] leads to slow decoding of CAA codons, inducing a translational pause that allows the P-site peptidyl-tRNAPro[cmo5UGG] to slip into the +1 frame. We have characterized 108 independent frameshift suppressor mutants in the gene encoding tRNAPro[cmo5UGG]. The altered tRNAs are still able to read all four proline codons in the A-site, but induce frameshifts after translocation into the P-site. Some of the mutations are in regions of the tRNA that are involved in interactions with components of the P-site. We hypothesize that the ribosomal P-site keeps a “grip” of the peptidyl-tRNA to prevent loss of the reading frame.

The structural basis for the fidelity of translation was modeled using computational methods. The flexibility of tRNA was explored using molecular dynamics and making a database of all crystallographic structures for tRNA. The modes of flexibility were compared based upon several metrics. A method for fitting cryo-EM with crystallographic structures was developed (MdMD), and also, for finding pathways between cryo-EM states. Biasing methods in molecular dynamics were used to model the pathway for the proofreading step of ribosomal translation. Atomic models were made for the Pre- and Post- accommodation state of the ribosome. These results indicated a new hypothesis for the mechanism of proofreading. There was no evidence for an induced fit mechanism in the large or small subunit of the ribosome during this step. The tRNA has a differential deformation in the kink during decoding that is based upon whether the tRNA is cognate or near-cognate. This difference in stored energy affects the outcome of proofreading, and the simulations of this step show that the ribosome presents some barriers, which would reject tRNA with insufficient stored energy.

Thesis (Ph.D.)--McMaster University (Canada), 1995. / Source: Dissertation Abstracts International, Volume: 56-12, Section: B, page: 6729. Adviser: J. P. Capone.

Thesis (Ph.D.)--McMaster University (Canada), 1995. / Source: Dissertation Abstracts International, Volume: 56-12, Section: B, page: 6730. Adviser: J. P. Capone.

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xv, 139 p.; also includes graphics (some col.) Includes bibliographical references (p. 123-139).

Thesis (Ph. D.)--University of Oregon, 1999. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 90-101). Also available for download via the World Wide Web; free to University of Oregon users. Address: http://wwwlib.umi.com/cr/uoregon/fullcit?p9947978.

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.

The transcription efficiency of transfer RNA genes is modulated by sequences contained in their 5'-flanking region. For a tRNA val₄ gene a pentanucleotide with the sequence TCGCT was identified between positions -33 and -38. I have previously proposed that this sequence may be involved in specifically determining the rate of transcription of this gene. A general form of this sequence, TNNCT was found associated with other Drosophila tRNA genes which showed high ill vitro transcription efficiency. To further elucidate the role of TCGCT in tRNA transcription, single and double base-pair changes were created in the sequence TCGCT using site-specific mutagenesis. Mutations in the nucleotides -38T, -35C and -34T showed decreased levels of transcription whereas nucleotide changes at the nucleotides -37C and -36G did not reduce template activity. Therefore the sequence which modulates transcription of the tRNAVal₄ gene does have the general form TNNCT. Competition experiments between the Val₄ mutant -38G.-35A and a tRNASer₇ gene showed the TNNCT mutant to be a better competitor for transcription than the wild type template. Experiments analyzing the time-course of transcription, the effects of temperature and the effects of ionic strength indicated that TNNCT was not involved in determining the efficiency of stable complex formation. It is proposed that the pentanucleotide is probably responsible for influencing the rate of initiation of transcription. A sequence TGCCT contained in the anticodon stem/loop region of the Val₄ gene was also mutagenized and shown to be involved in complex stability or the elongation of Val₄ tRNAs. Using deletion analysis of the 5'-flanking sequences of a tRNASer₇ gene, a second positive transcription regulatory element was delimited. This sequence was also found in the 5'-flanks of the tRNAVal₄ and a tRNAArg gene. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

The nucleotide sequence of the single Drosophila meianogaster tRNA gene contained in the recombinant plasmid, pDtl20R was determined by the Maxam and Gilbert method. This plasmid hybridizes to the 90 BC site on the Val Drosophila polytene chromosomes, a minor site of tRNA4 hybridization. The Val nucleotide sequence of the tRNA4 gene present in pDtl20R differs at four Val positions from the sequence expected from that of tRNA4 . The four differences occur at nucleotides 16, 29, 41 and 57 in the coding region. Comparison of the DNA sequence of pDtl20R to that of the plasmid pDt92R, which also hybridizes to the 90 BC site, indicates that the Drosophila fragments contained in these two plasmids are either alleles or repeats. The implications of these findings are discussed. An in vitro transcription system was developed from a Drosophila Schneider II cell line. This homologous cell-free extract support specific and accurate transcription of various Drosophila tRNA Val genes. The major product of transcription is a tRNA precursor which is processed to a tRNA sized species. Transfer RNA valine genes originating from different sites on the Drosophila chromosomes are transcribed at different rates. Comparison of the sequences in the internal promoter regions of the various genes indicates that the few differences within the coding regions may not be responsible for the observed difference in the rates of transcription. This conclusion is substantiated by studies with hybrid genes constructed during the course of this work. Preliminary evidence indicates that the Val tRNA gene which is transcribed at the highest rate may be preceded in its 5'-flanking region by a positively modulating sequence. Val The precursor RNAs directed by various tRNA genes are also processed at different rates. Transcription and processing experiments with hybrid genes suggest that nucleotide changes within the coding region, which do not affect the rate of transcription, influence the rate of processing. Time course and competition experiments demonstrate that at least two kinetic steps are required for the formation of a stable transcription complex. Studies with an in vitro constructed mutant missing in nucleotides 51-61 in the tRNA coding region suggests that this deleted region (which is highly conserved in eukaryotic tRNAs) may be involved in the primary interaction required for tRNA gene transcription. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate