Translation of picornavirus RNA takes place by internal initiation, determined by the presence of an internal ribosome entry site (IRES) in the 5'-untranslated region of the genomic RNA. Efficient translation from the human rhinovirus-2 (HRV-2) IRES is dependent on host cell <I>trans</I>-acting factors. These include unr, p38 and polypyrimidine tract binding protein (PTB). This thesis details the investigation into how these factors act to promote translation from the HRV-2 IRES. Unr, an RNA-binding protein with five cold-shock domains (CSDs), binds to the HRV-2 IRES and this interaction was studied by crude and then fine mapping of the binding sites of unr on the IRES. The functions of the CSDs of unr were investigated by point mutation of each of the CSDs and testing the ability of these mutants to bind the IRES and stimulate translation from it. p38, a WD-motif protein with no RNA-binding activity, was expressed using recombinant baculovirus-infected insect cells. An <I>in vivo</I> interaction between unr and p38 was demonstrated, and the effect of p38 on unr's binding to the HRV-2 IRES was tested <I>in vivo. </I>After gaining insight into the complexes of unr and p38 that form on the IRES, the function of p38 in translation from the HRV-2 IRES was demonstrated. Unr and PTB were also used as tools to compare the factor requirements of the HRV-2 and poliovirus IRESs for efficient translation. Finally, an investigation was made into the cellular role of unr, in terms of the cellular mRNAs that unr binds, and those whose translation it stimulates.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:596963 |
| Date | January 2001 |
| Creators | Brown, E. C. |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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