The aims of this grant are focused on a molecular dissection of the mechanisms by which yeast Ty1 elements replicate their nucleic acids, how they integrate newly made Ty1 cDNA into very specific genomic target regions, and how host functions participate in these processes. Replication of Ty1 elements is mediated by host RNA polymerase II, the Ty1-encoded reverse transcriptase (RT) enzyme, and a cellular tRNA primer. Based on our extensive mutational analyses, we will probe the RNA structures that appear to be required for retrotransposition, and how they interact with the reverse transcriptase, the primer tRNA, and the packaging machinery. Integration of the resulting cDNA is mediated by the Ty1-encoded integrase. The process of integration is targeted to very specific regions of the host genome, namely """"""""integration windows"""""""" of several hundred base pairs immediately upstream of RNA polymerase Ill-transcribed genes. We seek to understand how this targeting is directed, presumably by a combination of Ty1-encoded and host functions. We will carry out genetic analyses of Ty1 retrotransposon and host functions, supplemented by biochemical studies that exploit the in vitro systems we have previously developed for the study of Ty1 reverse transcription and integration. Finally, we will explore the idea that elevating intracellular Mn+2 might interfere with HIV-1 replication. Based on the results of these experiments, we will pursue the isolation of compounds that raise intracellular Mn+2 in human cells by interfering with the human Pmr1p protein.
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