Functional Analysis of TEV Internal Ribosome Entry Site

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A major deviation from the classical model of translation occurs for those mRNAs that lack a cap structure. These mRNAs possess a structured, non-coding region of 5' RNA known as an IRES (internal ribosome entry site). IRES are also beginning to be identified in cellular RNA. Generally, they are found under conditions where overall translation is compromised. The 5' untranslated region (UTR) of viral mRNA is sufficient to confer its cap-independent translation, however the mechanism of IRES translation (including viral translation) is not well understood. This project will elucidate the mechanisms of IRES function by using the 5' UTR of tobacco etch virus (TEV) as a model system. The TEV 5' UTR secondary structure has been mapped by chemical probing and mutagenesis. Three inter-related phases of the initiation process will be examined. These are 1) eIF binding, 2) helicase activity and 3) ribosome binding activity. Each of these activities will be measured for a select library of TEV constructs with differing translational activity. The eIF binding will be monitored by direct fluorescence assays. Base pairing changes associated with helicase activity will be elucidated by using fluorescent labels incorporated into specific locations in the RNA and nmr measurements. Ribosome binding will be measured by direct fluorescence methods and sucrose gradient analysis. The experiments described here will elucidate the molecular interactions that allow viral IRES to compete effectively with the much higher concentrations of cellular RNA for translation. Plant viral diseases affect a significant number of food crops world-wide and can have severe impact both on economic conditions and food supply. There is potential to use information from this research not only to control viruses but also to develop systems to produce desired proteins which are nutritionally beneficial, antibodies or have economic uses. TEV is one of the simplest IRES elements, so it serves as a model system to increase our understanding of more complex viral and cellular IRES function. The experiments will provide excellent training for students from high school through graduate school in both biochemical and quantitative measurements. Many of the students involved in research in the past have been from underrepresented minority groups and this is likely to continue in the future. These students will contribute to a diverse and well trained workforce in science and technology.