APPROVED Queuine is a bacterial metabolite that is salvaged by almost all eukaryotic species including algae, yeast, fungi and metazoans and is incorporated into transfer RNA for the amino acids asparagine, aspartic acid, histidine and tyrosine. The enzyme that carries out this reaction is a complex of two proteins, human queuine tRNA ribosyltransferase QTRT1 and QTRT2, referred to as the queuine insertase (QI) complex. Initial efforts in this thesis describe the steps to over-express the subunits of the QI complex individually and in combination in E. coli and the various strategies used to successfully remove the tag sequences from both proteins. The results of these efforts allowed the production of large amounts of catalytically active enzyme that facilitated much of the later studies. Importantly, the ability to purify the subunits independently allowed the isolation of protein that was free of bound tRNA substrate, which had repercussions for the successful outcome of subsequent work. Previously, Kelly?s laboratory reported on novel compounds capable of reversing disease progression in an animal model of Multiple Sclerosis. Using the recombinant QI complex and in vitro synthesized transfer RNA, this thesis provides a comprehensive examination of novel nucleobase substrates for the QI enzyme. It was found that the enzyme has an unexpectedly large substrate preference that is of particular relevance to drug development efforts. To help further inform drug design and substrate preference, kinetic studies were performed on the QI complex showing that nucleobase substrates bind to the enzyme before tRNA in accordance with an ordered sequential mechanism. Unexpectedly, unlike guanine?a non-physiological QI substrate?the natural queuine substrate was found to not follow normal Michaelis-Menten kinetics. Instead, both kinetic investigations and binding studies, evaluated by equilibrium dialysis, showed queuine acts as a homotypic positive allosteric regulator of the enzyme. Previously published protein purification studies identified a number of potential QI interacting partners. In an attempt to identify other interacting proteins, CRISPR technology, together with SmartFlare probes was employed to insert and select for a tag sequence integrated in the genome of MDAMB- 231 cells that would form a C-terminal purification-tag on the QTRT1 protein. Successful isolation and characterization of the QTRT1 genome modification was possible. Unfortunately however, one component of the dual-tag sequence was not appropriately recognized by the binding resin making the isolation of highly pure complex (in association with binding partners) impossible, an hence making the robust identification of QI interacting proteins uncertain.