ANALYSIS OF AN OLIGO(DA) OLIGO(DT) BINDING PROTEIN

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DESCRIPTION (Adapted from applicant's abstract): Critical control points in transcription can be governed by DNA binding proteins that recognize small numbers of targets within the genome. Studies of these specific trans-acting factors have led to insights into the regulation of many cellular processes including those that govern cell cycle progression, and normal and aberrant growth and differentiation. Relatively little is known about specific interactions of proteins with highly repeated DNA elements and the transcriptional influences that these interactions can exert. The simple nonalternating sequence dA.dT is highly repeated in many organisms. This sequence, which has been shown to adopt an unusual conformation, promotes transcription in yeast. It has been proposed that this transcriptional activation is related to the unique structure of dA.dT. The investigator recently reported the discovery of a new yeast DNA-binding protein that specifically interacts with nonalternating dA.dT. This protein (datin) has been purified, and its gene, (DAT1) cloned. More recent work shows that datin can negatively regulate the expression of genes which contain upstream dA.dT tracts. In addition, datin is able to positively regulate its own expression (datin's promoter contains dA.dT sequences). Since many yeast promoters contain dA.dT tracts, these observations suggest that datin can modulate the expression of large numbers of genes. These are two major objectives described in this proposal. First, the investigator will determine the molecular basis by which datin binds to dA.dT. Specifically the investigator will define the different forms of datin which exist in vivo, the precise DNA elements that these different forms of datin interact with, and the amino acid residues in datin which are necessary for this interaction. The investigator will also attempt to generate datin/oligo(dA).(dT) crystals for x-ray diffraction studies. Second, the investgator will determine what the physiological functions of datin are. Specifically, the investigator will determine the transcriptional regulatory effects that datin exerts on genes which contain upstream dA.dT tracts, and identify and characterize additional genes which encode functions that overlap the function of DAT1. These studies will not only contribute to the understanding of the molecular mechanisms responsible for specific DNA/protein interactions, but will also contribute to the understanding of the regulation of gene expression.