Transcription of protein-coding genes is one of the most fundamental processes that underlies all life and is a primary mechanism of biological regulation. The experiments included in this work are designed to provide new insights into the mechanisms of promoter escape, a critical, early event in the process of gene transcription by RNA polymerase II. The few initial steps in the process of mRNA synthesis that precede the completion of promoter escape are characterized by physical instability of the early RNA polymerase II transcription complex, formation of abortive transcripts, and strong dependence on ATP cofactor, downstream DNA, and the DNA-helicase activity of the general transcription factor TFIIH. Evidence recently obtained in the P.I.'s laboratory indicates that inhibitory interactions impede the forward movement of the polymerase and cause its promoter-proximal arrest. These interactions are overcome by the presence of an ATP cofactor, downstream DNA and the DNA-helicase subunits of the general transcription factor TFIIH. Dependence on all three cofactors reflects a common mechanism operating to facilitate promoter escape, and that this mechanism is continuously needed through the addition of the first 14-15 nucleotides to nascent RNA transcripts. At the transition between promoter escape and the elongation stage of transcription, it is expected that protein-protein and protein-DNA interactions will be disrupted or adjusted, leading to the release of some of the general transcription factors that are required for the early transcription complex. The focus of this project is to further explore this hypothetical view of early transcription by RNA polymerase II and its underlying mechanism. The studies include (i) tracking functional and physical changes in the early elongation complex, with particular emphasis on TFIIH and CTD phosphorylation, (ii) identifying polypeptides involved in inhibitory interactions during promoter escape, and (iii) determining the role of specific downstream promoter sequences in the efficiency of promoter escape. To pursue these goals, all the experiments will be carried out using the in vitro system assembled from highly purified polypeptide components, providing the controlled conditions in which the contributions of individual cofactors to rate-limiting steps can be evaluated. The experiments included in this work will enhance our understanding of the fundamental mechanisms of transcription that are applicable to all protein-coding genes in eukaryotic systems. The studies will also provide information necessary to identify possible targets for cellular regulatory mechanisms within the basal transcription machinery.
