While recent work has elucidated some of the basic apparatus and control elements of RNA polymerase II transcription, the structure and function of the DNA-protein template, chromatin, that this machinery operates on is not well understood. In addition to its recognized role of packaging DNA n the nucleus, chromatin can regulate gene expression. Chromatin mediated gene regulation is likely to play a role in developmental specific gene regulation and therefore will be important to understand both in terms of the normal development of an organism and in terms of aberrant processes such as carcinogenesis and genetic diseases. Although the structures of individual histones and nucleosomes are well described, the function and identity of other chromatin components is not understood. The goal of this proposal is to study a nonhistone, transcriptional regulatory protein that is likely to be a structural or regulatory component of chromatin. This protein, SPT5, is a yeast protein, thus both biochemical and genetic methods will be used in its study. Given the conservation of the components and details of the apparatus and regulation of transcription, the information and reagents gained from this study will be of use in studying similar processes in higher eukaryotes. Specifically, I propose to: first, use nuclease digestion studies to look for direct evidence that SPT5 can regulate chromatin structure. Second, biochemical interactions of SPT5 and chromatin or DNA will be assayed. Also, based on previous genetic data, biochemical interactions between SPT5 and two other proteins thought to interact with chromatin, SPT4 and SPT6, will be assayed. Finally a screen for gain of function mutations in the SPT5 gene will be performed; such mutants will allow genetic tests of models of SPT5 function.
