The long-term objective of this project is to understand how eukaryotic cells regulate gene expression at the level of transcription elongation. Eukaryotes regulate the expression of many genes including c-myc, c-myb, and c-fos by controlling the ability of RNA polymerase II to synthesize complete primary transcripts. Chromosomal rearrangements in Burkitt's lymphoma are associated with the derangement of c-myc's regulated block to elongation. The biochemical basis for regulating transcription elongation in animal cells is unknown and the RNA polymerase II elongation complex is poorly defined. It is unknown if any transcription initiation factors are incorporated into the complex after polymerase leaves the promoter region. THis proposal is a detailed characterization of the mammalian RNA polymerase II elongation complex. I have characterized a site in a eukaryotic gene that blocks transcription by RNA polymerase II and a factor (SII) that allows elongation through this site. RNA polymerase II elongation complexes will be assembled from rat liver general initiation factors which are free of elongation factors. A method has been developed to immunopurify arrested elongation complexes from in vitro transcription reactions using a monoclonal antibody against RNA. Isolated complexes resume transcript elongation when challenged with SII. The spectrum of initiation factors associated with the elongation complex will be identified. The mechanism of action and in vivo role of SII is unclear. Stable SII binding to an SII-responsive complex has not been observed. With our approach, we should be able to detect SII binding to the complex if binding has a long half-life. If the interaction is transient, we should be able to trap the SII-activated intermediate. This is an important first step in determining the biochemical basis for SII-mediated transcriptional activation. The size of the complex will be determined by nuclease footprinting experiments and electron microscopy in the presence and absence of elongation factor SII. The form of RNA polymerase II in an arrested elongation complex will be investigated. This work should facilitate our understanding of the control of transcript elongation by RNA polymerase II.
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