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Title: Analysis of ribonucleoprotein complexes using affinity purification in conjunction with mass spectrometry
Author: Nwokeoji, Alison Obinna
ISNI:       0000 0004 5358 0563
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2014
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Protein-protein/protein-RNA interactions are assuming increasing significance with the recent discoveries of the diverse and important roles RNA-protein complexes play in biological systems. As a result there is an increasing demand for high throughput analytical approaches for the analysis of these complexes. In this study, affinity purification-mass spectrometry (AP-MS) approaches are used to analyse protein-RNA complexes. Tandem affinity purification (TAP)-MS was used to analyse complexes associated with the Saccharomyces cerevisiae spliceosome and RNA processing pathways providing insight into the coupling of the various steps of gene expression. These approaches were also used to exhaustively distinguish two similar but functionally distinct Lsm complexes providing further insight into their mRNA processing pathways. These studies are consistent with the model that splicing occur co-trancriptionally and that several steps of gene expression is coupled to transcription. Utilizing HPLC-MS approaches in conjunction with other molecular biology techniques, the recently discovered Streptococcus thermophilus CRISPR/Cas complex was also investigated. The Cas protein complexes were identified and their stoichiometry determined using semi-quantitative data. This provided insight into the roles of these Cas proteins in the biogenesis of the CRISPR/Cas complex. Furthermore, the architecture of the CRISPR RNAs (crRNA) associated with the complex was determined, providing further insight into this mechanism of the crRNA processing in the novel Type III CRISPR/Cas complex.
Supervisor: Hornby, David P. ; Dickman, Mark J. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available