The DNA-dependent RNA polymerases play a central role in gene expression.
The aim of the proposed research is to contribute to a more precise genetic and biochemical description of the yeast RNA polymerase II. It is proposed that a combination of genetic and biochemical approaches are used in an effort to determine A) whether the polypeptides that copurify with RNA polymerase activity are all genuine subunits, B) whether other subunits exist which have thus far escaped biochemical detection and C) whether different forms of RNA polymerase II are responsible for the transcription of different kinds of genes. To accomplish these goals, the specific aims of the experiments outlined in this proposal are: 1) To identify and isolate the genes which specify each of the yeast RNA polymerase II subunits by probing a recombinant DNA expression library with antibodies and 2) To construct yeast mutants with lesions at defined RNA polymerase gene loci, resulting in conditional phenotypes, by using isolated subunit DNA sequences mutagenized in vitro to replace the wild type gene. The conditional mutants constructed through this procedure will be used in an effort to: A) identify genuine RNA polymerase II subunits through biochemical analysis, B) elucidate RNA polymerase subunit interactions and reveal previously undetected components of the enzyme through the isolation and analysis of extragenic suppressors, C) identify subunits through which specific regulatory factors act to control gene expression, and examine the possibility that there exist multiple forms of RNA polymerase II, by analyzing the effects of the mutant alleles on the relative ability to express specific genes. The health relatedness of this project derives from its contribution to the understanding of the basic molecular mechanisms which control gene expression.
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