Characterization of cap independent translation in potato virus Y.

摘要

Canonical translation initiation in eukaryotes takes place via cap dependent mechanism where translation initiation machinery assembles around the 5' cap of the mRNA. However, the uncapped RNAs of many viruses direct cap independent translation initiation mediated by cis acting sequences called internal ribosomal entry sites (IRES'). The characterization of an IRES in potato virus Y (PVY) is the topic of this dissertation. The results presented here establish that PVY contains an IRES that is distinct from any IRES' described to date.; In the first part of this dissertation, it is reported that PVY contains an IRES which is different from the previously described TEV IRES. Results from in vivo experiments using both mono- and bicistronic vectors show that the last 55 bases of the PVY 5'-UTR are sufficient to promote cap-independent initiation of translation through an internal initiation mechanism indicating that this region functions as an IRES.; In the second part of the dissertation, the minimal sequence required for PVY IRES activity was examined. Deletion analysis determined that this 55 base sequence contains two independent elements that cooperatively promote cap independent translation activity. The full IRES, containing both of these elements, exhibits activity which is greater than additive for these elements. Similarities and differences between the PVY IRES and other IRES elements are discussed.; IRES mediated translation initiation of cellular mRNAs has been somewhat controversial. Much of the controversy over IRES elements arises from the methods used to identify and characterize cap independent translation activity. Moreover, no system described to date is fully satisfactory. The final part of the dissertation describes a novel in vivo system for IRES analysis and its use to confirm the cap independent nature of translation enhancement by the recently characterized IRES from PVY. Biochemical analysis confirmed that transcripts generated from the Pol I promoter were uncapped. Inclusion of the PVY IRES element upstream of the reporter gene resulted in reporter gene activity, demonstrating that the IRES promoted translation from the uncapped message. To the best of our knowledge, this is the first time cap independent translation has been analyzed in vivo using a cap independent system.