RNA polymerase II catalyzes the synthesis of mRNA precursors in the nucleus of eukaryotic cells. The initiation of new RNA chains by RNA polymerase II is closely controlled and is believed to provide and important mechanism for the regulation of gene expression in response to environmental and physiological stimuli. The main focus of the proposed work is to better define the enzymatic steps involved in the initiation of transcription and to determine how promoter-specific factors alter the initiation process. Proposed work includes: 1. Continuation of studies with the SV40 major late promoter. although this promoter is recognized efficiently by the host cell transcription machinery, it lacks a TATA box and other consensus sequences usually associated with promoters transcribed by RNA polymerase II. Mutagenesis has shown that transcription is dependent on three elements, one of which is downstream of the transcriptional state site. Host proteins required for activity of the SV40 major late promoter will be identified. In order to better understand the mechanism of transcriptional initiation, the specific kinetic step or steps that are affected by mutants in each of the three promoter elements will also be determined. 2. Further development of a system for rapid purification of the enzymes required for promoter-directed initiation of transcription. The method involves assembly of a stable preinitiation complex on a template DNA bound to a solid support. After stringent washing to remove unbound proteins, initiation is allowed to proceed in situ by addition of nucleoside triphosphates. This system will be used to study the catalytic events that occur early in RNA synthesis, using both the SV40 major late promoter and promoters of more conventional structure. Intrinsic initiation rate will be measured as a function of nucleoside triphosphate concentrations, and the specific role of hydrolyzable ATP or dATP in the reaction will be determined. 3. Studies to determine why multiple transcription factor binding sites are required for the activity or promoters transcribed by RNA polymerase II. These studies will involve characterization of reaction kinetics and protein binding, using synthetic promoters with different arrangements of recognition sites for the transcription factor Sp1. Although Sp1 appears to bind to each site independently in these model promoters, activation of transcription is known to be highly cooperative.
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