Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Stable RNAs undergo a wide variety of post-transcriptional modifications, that add to the functional repertoire of these molecules. Some of these modifications are catalyzed by stand-alone protein enzymes, while some others are catalyzed by RNA-protein complexes. tRNAs from all domains of life contain many such modifications, that increase their structural stability and refine their decoding properties. Certain regions of tRNAs are more frequently modified than others. Two such regions are the anticodon loop, and the TψC stem. In the halophilic euryarchaeon Haloferax volcanii, tRNATrp and tRNAMet, both of which are transcribed as intron-containing pre-tRNA forms, contain Cm34 and ψ54, in addition to other modifications, in these two regions, respectively. The Cm34 modification in both cases is RNP-mediated: tRNATrp Cm34 formation being guided by its own intron, while that of tRNAMet being guided by a unique guide RNA called sR-tMet. We created genomic deletion of H. volcanii tRNATrp intron by homologous recombination based technique, and showed that this strain is viable, and does not demonstrate any observable growth phenotype. However, the corresponding modifications are absent in this intron-deleted strain. Our structural and functional characterizations of sR-tMet revealed that it is unique in its structural properties and deviates considerably from its homologs in other Archaea. We also identified a novel L7Ae (a core protein associated with archaeal methylation guide RNPs) binding motif in sR-tMet. ψ54, the near universal modification found in TψC stem-loop of archaeal tRNAs is catalyzed by the protein Pus10. An earlier study from our laboratory had shown that Pus10 from two different archaea, Methanocaldococcus jannaschii (MjPus10) and Pyrococcus furiosus (PfuPus10) have differential activities towards ψ54 formation. Using the crystal structure of Human Pus10 as template, we created homology models of MjPus10 and PfuPus10 proteins and identified several residues and motifs that might lead to this difference in activity. By a combination of both in vitro and in vivo mutational approaches, we confirmed several previously unidentified residues/motifs that serve as positive determinants of tRNA ψ54 formation. Finally, as an extension to this study, we have identified a novel tRNA ψ54 forming activity in mammalian nuclear extracts, and attributed this activity to Pus10.

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Several Saccharomyces cerevisiae mutant tRNAQ2 species (glutamine isoacceptor, CUG anticodon) were synthesized and assayed for aminoacylation activity with Saccharomyces cerevisiae glutaminyl-tRNA synthetase. The derived steady state parameters were compared to similar datasets from the literature. The mutants behaved analogously to similar mutant species based on tRNA from Escherichia coli, but with slightly relaxed specificity as revealed by comparison of kcat/KM values relative to wild type in vitro transcribed tRNA. Additionally the eukaryotic N-terminal domain appendage, as found in Sce glutaminyl-tRNA synthetase, is considered in light of the discovery of non-canonical aminoacyl-tRNA synthetase functions, including its role in the assembly of the multiple aminoacyl-tRNA synthetase complex.

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Thesis advisor: Abhishek Chatterjee / Thesis advisor: Eranthie Weerapana / In nature, just twenty canonical amino acids are responsible for the creation of nearly all proteins. Genetic code expansion (GCE), or the incorporation of noncanonical amino acids (ncAAs) into living cells, is a powerful tool that expands the studies we are capable of performing using proteins. This technology relies on engineered aminoacyl-tRNA synthetase (aaRS)/tRNA pairs that are orthogonal to the host cells’ endogenous aaRS/tRNA pairs, and one of the main limitations of GCE arises from the inefficiency of these suppressor tRNAs when expressed in a foreign host cell. To address this limitation, we have previously reported a strategy for the virus-assisted directed evolution of tRNAs (VADER) which is uniquely capable of addressing the specific needs of tRNA evolution. In order to advance the capabilities of VADER, we made a number of modifications to the VADER selection scheme. First, we designed and executed a modified VADER selection that enabled the evolution of a new class of tRNAs, and with this VADER selection, we were able to generate a first-generation E. coli tyrosyl tRNA (tRNATyr) variant that was three times as active as its wild-type equivalent. Next, we introduced a number of refinements to the VADER strategy to generate VADER 2.0, an improved workflow capable of screening larger libraries and libraries encoding more active variants. Using VADER 2.0, we created second-generation tRNAPyl and tRNATyr mutants that achieved incorporation efficiencies that were greater than five-fold higher than their wild-type equivalents across a wide variety of substrates, enabling exciting GCE experiments that would not be possible otherwise. / Thesis (PhD) — Boston College, 2023. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

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