The aims of this grant are focused on a molecular dissection of the mechanisms by which yeast Tyl elements replicate their nucleic acids, how they integrate newly made Tyl cDNA into very specific genomic target regions, and how host functions participate in these processes. Replication of Tyl elements is mediated by host RNA polymerase II, the Tyl-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 Tyl-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 Tyl-encoded and host functions. We will carry out genetic analyses of Tyl retrotransposon and host functions, supplemented by biochemical studies that exploit the in vitro systems we have previously developed for the study of Tyl 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 Pmrlp protein.
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