The long-range goal of this research is to understand, at the molecular level, how transcription of eukaryotic protein-coding genes is initiated and regulated. The goal of the research described in this proposal is to determine the enzymatic mechanism of transcription initiation catalyzed by RNA polymerase II and a set of five essential initiation factors that have been purified from liver. These factors direct transcription initiation from the core regions of many promoters, including those of viral, lymphokine, and liver genes. Rat liver has been chosen as a model for these studies because the initiation factors can be purified to homogeneity from rat liver in quantities sufficient for serious biochemical analysis of their structures and mechanisms of action. The overall aims of this proposal remain essentially the same as those described in the previous proposal: to reconstitute in vitro, with purified enzymes, accurate initiation of transcription of eukaryotic protein-coding gene and to use this reconstituted system to elucidate the biochemical mechanism of initiation by RNA polymerase II. The research will be organized along the following lines: (1) Detailed characterization of the structure and activities of the essential factors. (2) Development of procedures for purification of a high molecular weight, endogenous TATA-factor. (3) Investigation of the mechanism by which ATP activates initiation. (4) Detailed analysis of the interactions of RNA polymerase II and the initiation factors with each other an with DNA during assembly of the preinitiation complex. These studies should provide a foundation for future analysis of the mechanisms by which transcription of eukaryotic protein-coding genes is regulated. They should also yield information about the expression of a large class of genes that have biomedical importance.
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