The long term goal of this proposal is to increase our understanding of the specific mechanisms by which RNA polymerase II overcomes the barrier to elongation imposed by chromatin, and also to understand how these mechanisms are used to regulate transcription. Specifically, this proposal concerns two proteins, Spt4 and Spt5, from the budding yeast Saccharomyces cerevisiae. Spt4 and Spt5 are non-histone transcriptional regulatory proteins that are thought to function by affecting chromatin structure. Recent genetic and biochemical studies of Spt4 and Spt5, and of their human homologs, have provided strong evidence that these proteins are regulators of transcription elongation. In one case, the human Spt5 protein was found to be required for the function of the HIV Tat protein, which regulates the rate of transcription elongation on the HIV genome. Based on these data, it has been proposed that Spt4 and Spt5 form a protein complex that assists RNA polymerase II to transcribe through the barrier to elongation imposed by nucleosomes. Furthermore it was proposed that this Spt4/Spt5 complex helps to maintain the chromatin structure of transcribed genes by restoring normal nucleosomal structures after polymerase has transcribed over a DNA sequence.
The Specific Aims of this proposal are: First, to investigate the association of the Spt4/Spt5 complex with chromatin and determine if alteration in Spt4/Spt5 function leads to disruption of chromatin. These experiments will use the technique of chromatin immunoprecipitation to monitor the association of Spt5 with chromatin and assays of nuclease accessibility to monitor chromatin structure in wild type and mutant cells. Second, to investigate the previously observed association of the Spt4/Spt5 complex with RNA polymerase II and determine how other factors play a role in that association. These studies will use a coimmunoprecipitation assay to monitor the association of Spt5 and RNA polymerase II. Also, other factors that interact with the Spt4/Spt5 complex will be identified by immunoaffinity purification. Third, to study Chd1, a protein that is a component of a chromatin remodeling complex, contains chromodomains (a protein domain thought to mediate chromatin functions), and that interacts with Spt5. Also, this Specific Aim includes the analysis of genetic suppressors of previously identified spt5 mutations. These experiments should yield new insights into the mechanisms and regulation of transcription elongation in the context of chromatin. Furthermore, these insights into Spt4/Spt5 function should provide information relevant to understanding the transcriptional regulation of HIV.
Showing the most recent 10 out of 13 publications
