The synthesis of mRNA in eukaryotic cells is catalyzed by RNA polymerase II. The overall objective of this proposal is to relate the complex molecular structure of mammalian RNA polymerase II to its ability to catalyze specific steps in the transcription reaction. Mammalian cells contain two forms of RNA polymerase II, designated IIO and IIA, that differ with respect to the level of phosphorylation within the C- terminal domain of their largest subunit. The C-terminal domain consists of 52 repeats of the consensus sequence tyr-ser-pro-thr-ser-pro-ser and is heavily phosphorylated in RNA polymerase IIO and unphosphorylated in RNA polymerase IIA. The reversible phosphorylation of this domain is thought to play a role in the transition of RNA polymerase II from the initiation to the elongation phase of transcription. This proposal is specifically concerned with understanding the functional significance of the repetitive C-terminal domain and the consequences of its phosphorylation. The specific objectives are as follows: 1) determine the level of phosphorylation of the C-terminal domain of subunit IIa as a function of the position of RNA polymerase II in the transcription cycle, 2) purify and characterize the protein kinases involved in the phosphorylation of this domain, 3) purify and characterize the phosphoprotein phosphatases involved in dephosphorylation of this domain, 4) determine the relationship between factors involved in the phosphorylation of this domain and essential transcription factors, and 5) examine the consequences of pertubating the level of phosphorylation on the ability of RNA polymerase II to catalyze specific steps in the transcription reaction. The experiments proposed here will provide a critical test of the hypothesis that each transcription cycle involves the reversible phosphorylation of the C-terminal domain of RNA polymerase subunit IIa and that the phosphorylation of this domain is subsequent to the interaction of RNA polymerase with the promoter. The existence of such a cycle has important implications with respect to both the basic mechanism of transcription and the regulation of gene expression.
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