Abstract
Translation of mRNA into protein represents the final step in the gene-expression pathway, driving the formation of the proteome from genomic information. The regulation of this process is a mechanism that is used to modulate gene expression in a wide range of biological situations. Protein synthesis is principally regulated at the initiation stage, allowing for rapid, reversible control of gene expression. Progress over recent years in determining the structures and activities of regulatory factors, and in mapping their interactions, have advanced our understanding of the complex translation initiation process. These developments have provided a solid foundation for studying the regulation of translation initiation by mechanisms that include the modulation of initiation factor activity, internal ribosome initiation and through sequence-specific RNA-binding proteins. This thesis focused on translational control during viral infection, where we investigated the role of Severe Acute Respiratory Syndrome non-structural protein 1 and Enterovirus 71 Internal Ribosome Entry Site in this process. To establish the function of SARS NSP1 protein in translation regulation we attempted the identification of NSP1 protein partners using several types of protein affinity chromatography. Using a wide range of approaches, we could not detect nor confirm the association of NSP1 with any cellular proteins. To dissect the role of FBP2, we engineered a wide range of recombinant FBP2 proteins of different lengths and analysed their interactions with IRES elements using biochemical techniques. This allowed us to characterize the interaction of EV71 IRES with initiation factors eIF4A, eIF4E, eIF4G and FBP2. Finally, we used chemical probing of RNA structure in solution to establish the secondary structure of the BiP IRES. We identified the formation of a structured RNA scaffold of 220 nucleotides comprising 3 major domains.