Thesis (Ph.D.) - University of Queensland, 2005. / Includes bibliography.

N6-threonylcarbamoyl adenosine (t6A) is a universally conserved tRNA modification found at position 37 of tRNAs which decode ANN codons. Structural studies have implicated its presence as a requirement for the disruption of a U-turn motif in certain tRNAs, leading to the formation of properly structured anticodon stem loop. This structure is proposed to enhance the base pairing between U36 of tRNA and A1 of the codon which aids in translational frame maintenance. Despite significant effort since its discovery in the 1970s the enzymes involved in its biosynthesis remained undiscovered. Bioinformatic analysis identified two proteins as likely candidates for t6A synthesis, YrdC and YgjD. Subsequent gene knockout experiments in yeast were consistent with their involvement in t6A biosynthesis in vivo. Furthermore, clustering between the bacterial genes ygjD, yeaZ and yjeE as well as the identification of a protein interaction network between YgjD, YeaZ, and YjeE suggested that YeaZ and YjeE might be involved in t6A biosynthesis. The genes encoding ygjD, yeaZ, yrdC and yjeE were cloned from E. coli and the recombinant protein was purified. Experiments analyzing the incorporation of [U-14C]-L-threonine and [14C]-bicarbonate (substrates previously indicated in its biosynthesis) into tRNA in the presence of these four proteins demonstrated the first reconstitution of the t6A pathway in vitro. LC-MS analysis verified the formation of t6A, and these proteins were renamed TsaD (YgjD), TsaB (YeaZ), TsaC (YrdC), and TsaE (YjeE). Biochemical characterization of this pathway suggested that the formation of t6A proceeds through an unstable threonylcarbamoyl adenosine monophosphate (TC-AMP) intermediate, which is produced by TsaC from its substrates CO2, L-threonine and ATP. To investigate this reaction in more detail a coupled assay was developed, enabling sensitive detection of turn over. TsaC is a promiscuous enzyme which readily accepts several amino acids as substrates. The formation of t6A from TC-AMP is catalyzed by TsaD, TsaB, and TsaE. Of these three proteins only TsaD is universally conserved suggesting it is the protein catalyzing the transfer of the threonylcarbamoyl moiety to A37 of tRNA. This transfer is not promiscuous as only TC-AMP serves as an efficient substrate for t6A formation. Structural investigation of these proteins are consistent with the formation of a single protein complex potentially alleviating issues with the reactivity and instability of TC-AMP.

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.

RNA binding proteins (RBPs) play crucial roles in regulating every stage of the mRNA life cycle and mediating non-coding RNA functions. Despite their importance, the specific roles of most RBPs remain unexplored because we do not know what specific RNAs most RBPs bind. Current methods, such as crosslinking and immunoprecipitation followed by sequencing (CLIP-seq), have expanded our knowledge of RBP-RNA interactions but are generally limited by their ability to map only one RBP at a time. To address this limitation, we developed SPIDR (Split and Pool Identification of RBP targets), a massively multiplexed method to simultaneously profile global RNA binding sites of dozens to hundreds of RBPs in a single experiment. SPIDR employs split-pool barcoding coupled with antibody-bead barcoding to increase the throughput of current CLIP methods by two orders of magnitude. SPIDR reliably identifies precise, single-nucleotide RNA binding sites for diverse classes of RBPs simultaneously. Using SPIDR, we explored changes in RBP binding upon mTOR inhibition and identified that 4EBP1 acts as a dynamic RBP that selectively binds to 5’-untranslated regions of specific translationally repressed mRNAs only upon mTOR inhibition. This observation provides a potential mechanism to explain the specificity of translational regulation controlled by mTOR signaling. SPIDR has the potential to revolutionize our understanding of RNA biology and both transcriptional and post-transcriptional gene regulation by enabling rapid, de novo discovery of RNA-protein interactions at an unprecedented scale.

published_or_final_version / abstract / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy

Thesis (Ph.D.) -- McMaster University, 2006. / Supervisor: David W. Andrews. Includes bibliographical references (leaves 110-117).

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2007 Aug 15.