Biochemical characterization of the human queine insertase complex
Citation:
AL-QASEM, MASHAEL ABDULLAH, Biochemical characterization of the human queine insertase complex, Trinity College Dublin.School of Biochemistry & Immunology.BIOCHEMISTRY, 2018Download Item:
Abstract:
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.
Sponsor
Grant Number
King Abdullah for external scholarship, The Ministry of Higher Education, Kingdom of Saudi Arabia
Author's Homepage:
http://people.tcd.ie/alqasemmDescription:
APPROVED
Author: AL-QASEM, MASHAEL ABDULLAH
Advisor:
Kelly, VincentPublisher:
Trinity College Dublin. School of Biochemistry & Immunology. Discipline of BiochemistryType of material:
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Full text availableKeywords:
QTRT1, QTRT2, QI COMPLEX, QI ENZYMEMetadata
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