The major research goal of this proposal is to understand the function of proteins that have very general roles in transcription initiation by RNA polymerase II in vivo. Because transcription initiation is usually the major regulatory step in differential gene expression, it is fundamental that the mechanisms involved in transcriptional regulation are understood in detail. Our approach has been to use a genetic selection to identify mutations that increase the activity of a basal promoter in yeast, with the expectation that these mutations would identify proteins that affect the activity of the general transcription factors or pol II in vivo. We have identified six genes, designated BUR1 through BUR6 (BUR = Bypass UAS Requirement) using this selection. The pleiotropic phenotypes of the bur mutations, combined with other genetic and molecular analysis, indicate that they have fairly general effects on transcription. The BUR genes have all been cloned and sequenced, and they have been found to encode a number of known proteins with direct biochemical roles in transcription, including histones H2A and H2B, histone H3 (BUR5), and MOT1 (BUR3), a direct ATP-dependent inhibitor of the TATA-binding protein. In addition, the cloning of two other BUR genes revealed significant homology to previously identified proteins of known biochemical function. The N- terminal half of BUR1 is 67% homologous to CDC2/CDK, and is therefore likely to encode a protein kinase that phosphorylates some key transcription factor. BUR6 encodes a protein with significant homology to histone H2A that is likely to be either a new histone variant or a novel H2A-related protein. We propose a number of genetic and biochemical approaches to: 1) identify and characterize the substrates and regulators of the putative BUR1 kinase, and 2) determine whether BUR6 is an authentic histone H2A variant and how bur6 mutations affect transcription. The results of these studies should significantly advance our current understanding of the roles of protein phosphorylation and chromatin in transcription initiation. Furthermore, since many fundamental aspects of transcription have been conserved throughout the evolution of eukaryotes, these studies in S. cerevisiae serve as a useful model system for understanding transcription factors and regulatory mechanisms that are common to all eukaryotes.
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