Transcriptional regulation is a primary target of many signaling and developmental pathways and regulation of transcription is one of the key steps in control of cell growth, differentiation and development. Published studies suggest at least two classes of core promoters that differ in whether they contain a consensus TATA element and in their requirements for the coactivator complexes TFIID and SAGA. Available evidence suggests that these promoter types differ in the mechanism of interaction with the basal factors, in regulation by chromatin and its modifications, and in their response to transcription regulators. Since most mechanistic studies to date have been conducted on TATA-containing promoters, little is understood about the mechanisms of initiation and regulation at the ~80% of genes lacking TATAs, leaving a large gap in understanding how most RNA polymerase (Pol) II transcribed genes are regulated. The long term goals of this project are to determine the mechanisms of RNA Pol II transcription initiation and how these mechanisms are utilized as targets for gene regulation. The rationale for this work is that determining the mechanisms used in initiation and its regulation will form the molecular basis for understanding defects in transcription disorders leading to many types of human disease. The objectives of this application are to determine the mechanisms utilized for initiation and regulation at TATA-less promoters, the specificity, function, and overlap of the coactivators TFIID and SAGA and the mechanism of DNA opening, transcription start site (TSS) scanning and TSS recognition during the dynamic process of transcription initiation. Our proposed research will utilize biochemical, molecular genetic, genomics, and biophysical approaches to examine transcriptional regulation in S. cerevisiae because the advantages in molecular genetics and biochemistry in this system allow rapid and precise mechanistic investigations. The basal machinery, TFIID and SAGA are all conserved so that results from the yeast system are directly relevant to human gene regulatory mechanisms. Recent findings and new approaches have called into question the initial TFIID and SAGA-regulated characterization of promoters and suggest a potentially large overlap between the sets of genes modulated by these factors. We will use a new approach we developed to map the genome-wide location, specificity and functional importance of these two coactivators on genome-wide expression. We will utilize a new in vitro system that transcribes TATA-less promoters to investigate biochemical mechanisms of these coactivators and how they are regulated. We will also use an innovative single molecule approach to investigate the dynamic mechanism of transcription initiation in the Pol II system at TATA-containing promoters. Our proposed research is significant because it will lead to a vertical advance in understanding the role of two conserved coactivators that together regulate nearly all Pol II transcription, illuminate mechanisms used at the major class of Pol II promoters, and reveal unstable intermediates and rate-limiting steps in the transcription initiation pathway.
Transcriptional regulation is one of the key mechanisms for control of cell growth, differentiation, and development, and defects in transcription directly contribute to many human illnesses. Understanding the mechanism of transcription and its regulation will form the basis for understanding the molecular defects in transcription disorders leading to many types of cancer, heart disease, neurological disorders, and birth defects. It will also provide the molecular basis for identifying new avenues of potential interventions and therapies in patients with these disorders that target specific and rate-limiting steps in gene regulation.
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