FACTORS INVOLVED IN TRANSCRIPTION BY RNA POLYMERASE II

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The goal of this proposed project is to come to a better understanding of the elongation phase of transcription by RNA polymerase II. The formation and maintenance of elongation complexes which are competent to synthesize mRNAs will be investigated. One of the major goals of the work is to characterize two recently identified factors that seem to be involved in controlling the formation of such complexes. One of these factors is tightly coupled to the preinitiation complex and forces the polymerase into an elongation mode in which only short transcripts can be formed. The second factor acts after initiation and allows the polymerase to proceed to the 3' end of the transcribed gene. An in vitro approach utilizing Drosophila Kc cell nuclear extracts will be taken for all of the proposed studies. DNA templates immobilized to paramagnetic beads will be used to manipulate transcription complexes formed on physiological promoters. Dissection of the processes leading to the formation of mature elongation complexes will be accomplished by changing the factors present in the reactions following magnetic concentration of the complexes. An examination of the properties of mature elongation complexes will determine how stably the elongation factors interact with the complex and if elongation complexes formed by initiation from different promoters have different properties. Another goal is to determine the biochemical details of the function of DmS-II, a Drosophila RNA polymerase II elongation factor. These studies will be aided by the availability of large amount of the intact protein and domains of the protein which have been produced in E. coli from the cDNA. The clear involvement of elongation control in the expression of numerous oncogenes and viruses including HIV, demands that the biochemical details of the process be elucidated so that rational approaches to controlling the diseased state may be made. The proposed studies should shed light on the mechanisms controlling elongation and, furthermore, will allow the formation of a more coherent picture of the complete transcription cycle.