The ultimate goal of this research is to understand how eukaryotic messenger RNA synthesis is regulated at the molecular level. An important first step will be to determine the enzymatic mechanism of initiation catalyzed by RNA polymerase II and a set of """"""""initiation factors"""""""" (termed a, b, d, e and g) purified from yeast. Those proteins are now available in quantities sufficient for mechanistic studies. Factor b fails to support transcription reconstituted with pure forms of polymerase and the other initiation factors, but a factor b """"""""holoenzyme"""""""" with that capacity has been isolated. Likewise, a """"""""native"""""""" polymerase- factor g complex with specific activity much higher than that of polymerase or factor g purified separately has been identified. These holoenzymes will be purified to establish a fully defined system capable of efficient template utilization. The initiation mechanism will be investigated through physical and enzymological studies. The structure and dynamics of protein-DNA assemblies will be determined by nondenaturing gel electrophoresis and chemical or enzymatic """"""""footprinting"""""""" methods. Single- and multiple-round transcription protocols will be used to test whether phosphorylation and dephosphorylation of RNA polymerase II are major determinants of reaction efficiency. Factor a, the yeast homologue of the human initiation factor TFIIE, will be mutagenized and purified from E. coli to determine if conserved domains in its primary structure are important for transcription activity. Temperature-sensitive mutants will be isolated and analyzed to determine if factor a/TFIIE is required for RNA synthesis in vivo.
