Studies of Elongation Arrest and Transcript Hydrolysis by RNA Polymerase II

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9604016 Hawley In recent years it has become increasingly clear that eukaryotic transcription is regulated not only before synthesis of the transcript is initiated, but also at the level of elongation of the RNA chains. Little is know about how this regulation is implemented or about the structure and function of RNA polymerase when it is in the process of elongating along the DNA template. This research is a biochemical analysis of the proteins and nucleic acid sequences that determine the transcription elongation behavior of human RNA polymerase II in vitro. Two proteins that alter the elongation properties of RNA polymerase II will be studied: TFIIF which increases the rate of elongation and SII which enables RNA polymerase II to read through DNA arrest sites that would otherwise block it progression. The structure and function of arrested and productive elongation complexes will be investigated with the goal of obtaining mechanistic information about how arrest occurs at arrest sites (which share some features with better understood termination sites), and how TFIIF and SII interact with those complexes to prevent or relieve elongation arrest. Transcription complexes will be isolated and studied in vitro by attaching a DNA template consisting of a promoter and the adenovirus major late arrest site to agarose beads and assembling initiation, elongation or arrested complexes in an extract of nuclear proteins from human cultured cells. Such complexes were used previously to show that the base pairing of an oligonucleotide to the RNA in transcription complex has a profound effect on the behavior of the complex depending on where the base-pairing occurs. This work led to the hypothesis that arrest results in a region of the nascent RNA being pulled back into the polymerase, and will be further examined by analyzing the relationship between oligonucleotide hybridization position and the conformation of the RNA polymerase at the arrest site. Other experiments will map the upstream and do wnstream boundaries of the polymerase on the DNA template in arrested and productive elongation complexes, and will investigate the contribution of the surrounding DNA sequence and the mechanism of TFIIF stimulation by measuring the rate of single nucleotide incorporation into the RNA chain at defined positions on the DNA template. This research will provide a detailed view of the structure of RNA polymerase II elongation complexes and will suggest possible mechanisms by which these complexes can be regulated. In recent years it has become increasingly clear that eukaryotic transcription is regulated not only before synthesis of the transcript is initiated, but also at the level of elongation of the RNA chains. Little is known about how this regulation is implemented or about the structure and function of RNA polymerase when it is in the process of elongating along the DNA template. This research is a biochemical analysis of the proteins and nucleic acid sequences that determine the transcription elongation behavior of human RNA polymerase II. This research will provide a detailed view of the structure of RNA polymerase II elongation complexes and will suggest possible mechanisms by which these complexes can be regulated.