The long-term objectives of the proposed research are to identify factors that play important roles in transcription by RNA polymerase II (pol II) and to determine the mechanisms by which these factors regulate this process. This application focuses on transcription elongation and a recently discovered elongation factor in Saccharomyces cerevisiae, the Paf1 complex. Previous work showed that the Paf1 complex interacts with RNA pol II and transcription elongation factors, associates with the coding regions of genes, and recruits chromatin modifying proteins to elongating RNA pol II.
The Specific Aims represent extensions of our previous studies on the Paf1 complex.
Specific Aim 1 is to investigate the requirement for the Paf1 complex in transcription elongation and RNA 3' end formation. The effect of the Pafl complex on RNA chain growth and RNA pol II progression will be measured by in vivo and in vitro elongation assays. The role of the Paf1 complex in RNA 3' end formation and transcription termination will be studied at polyA-dependent and polyA-independent genes through the analysis of mutant strains.
Specific Aim 2 is to address the mechanism by which the Paf1 complex associates with RNA pol II. The role of the RNA pol II C-terminal domain in Paf1 complex function will be investigated by genetic and biochemical interaction studies, the step in the transcription cycle at which the Paf1 complex associates with RNA pol II will be determined by chromatin immunoprecipitation (ChIP) and in vitro transcription complex assembly assays, and the hypothesis that gene-specific regulatory proteins recruit the Paf1 complex to a gene will be tested by ChIP and northern analyses.
Specific Aim 3 is to investigate the functions of three proteins implicated by our genetic studies on the Paf1 complex to be involved in transcription elongation and chromatin-mediated regulation of gene expression. To elucidate their relationship to the Paf1 complex, these proteins, which represent members of a signal transduction cascade and a previously unstudied protein, will be analyzed by methods that include ChIP, genetic screens, and affinity chromatography. Since the proteins involved in transcription by RNA pol II are conserved throughout eukaryotes, the information that is learned from these studies in yeast will advance our understanding of transcription elongation in humans, where a disruption in this process leads to important human diseases including cancer and AIDS. ? ?
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