The long-term goal of this research is to understand the regulation of eukaryotic RNA synthesis at the molecular level. The general strategy to be employed involves transcription in cell-free systems. It is proposed to extend and improve the existing in vitro approaches in two ways. First, in order to define and characterize the macromolecular complex which is the target of regulatory events, a series of transcription complexes containing DNA template and RNA polymerase II will be prepared. Both newly-initiated and elongating complexes will be studied; in all cases template protection and RNA polymerase configuration and subunit composition will be examined. Studies will also be done to determine the subset of RNA polymerase II subunits involved solely in elongation. Second, in order to more faithfully reproduce in vivo transcription levels and patterns, more physiological templates than the currently-used purified DNAs will be prepared. In this part of the research, we will continue our current studies on reconstituted chromatin templates; we will also prepare chromatin templates from the cell in as close to their native state as possible. Recombinants between genes of interest and bovine papilloma virus will be made and propagated as episomes in mouse cells. Genes in such constructs continue to be properly regulated and may be recovered as minichromosomes. These purified episomes will be used as templates for in vitro RNA synthesis with the intent of demonstrating in vitro transcription levels proportional to the corresponding in vivo levels. Both metal-inducible (metallothione) and developmentally regulaterd (Beta-globin) promoters will be used in the constructs. For those cases in which in vivo transcription patterns can be duplicated, the minichromosomes will be examined for induction-correlated features. These would include: (i) the presence of regulatory proteins bound at the promoter, or at other known regulatory sites (enhancers), and (ii) changes in the configuration of the promoter (absence of nucleosomes, presence of denatured regions, etc.).
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