This is a application to continue a molecular genetic analysis of the mechanisms of Ty1 retrotransposition in Saccharomyces. Many of the structural features of Ty1 and its life cycle are similar to retroviruses. Ty1 has Long Terminal Repeats (LTRs) and encodes two overlapping ORFs (Open Reading Frame) analogous to the retroviral gag and pol genes. Ty1 utilizes reverse transcriptase and a tRNA primer for DNA synthesis from the viral RNA template and insertion into host chromosomal DNA is mediated by an integrase. Virus-like particles containing viral RNA are formed in the cytoplasm and are the site of DNA synthesis. They are intermediates in the Ty1 transposition process but, unlike the retroviruses, true viral particles are not released from the cells. This proposal utilizes the genetically tractable Saccharomyces Ty1 system to uncover fundamentals of the process of retrotransposition which are likely to impact on our understanding of retrovirus biology. In his earlier work, Dr. Boeke developed an in vivo assay for monitoring and/or selecting for transposition events in vivo. He used this system to identify and characterize host functions required for the retrotransposition process. This proposal outlines experiments designed to continue his characterization of the retrotransposition process of Ty1 with particular emphasis on identifying and exploring the function of host factors involved in the process. A detailed analysis of reverse transcription of Ty1 will be undertaken using genetic, molecular, and biochemical approaches. Their work has demonstrated that the primer tRNA sequence is not inherited by the progeny Ty1 DNA. Models are presented to explain this result involving strand transfer of less than full-length DNA intermediates, referred to as strong stop DNA. These models of the reverse transcription process will be tested. Virus-like particles were found to be enriched for tRNASerGCU and preliminary studies suggest a role in transposition. The function of this tRNA in the Ty1 life cycle will be determined and the structural basis of its efficient packaging into virus-like particles determined using molecular genetic approaches.
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