EUKARYOTIC TRANSCRIPTION COMPLEXES

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Transcription initiation at a eukaryotic protein-encoding gene involves assembly of a multiprotein-DNA complex containing RNA polymerase II, SRB/Mediator components, six general transcription factors (GTFs), and promoter DNA. The fully assembled transcription initiation complex contains more than 50 distinct polypeptides and has a molecular mass of more than 3 MDa. The proposed work addresses the structure of the transcription initiation complex, the pathway of assembly of the complex, and the promoter sequence elements that govern stability, assembly, and function of the complex. The proposed work has four specific aims: Specific Aim 1: To use systematic site-specific protein-DNA photocrosslinking to map protein-DNA interactions within the transcription initiation complex. Specific Aim 2: To use kinetic site-specific protein-DNA photocrosslinking to define the kinetics of formation of protein-DNA interactions within the transcription initiation complex. Specific Aim 3: To use binding-site selection to define promoter sequence elements recognized by GTFs. Specific Aim 4: To use high-density-array expression analysis to define the functional significance of promoter sequence elements recognized by GTFs. The results to be obtained will contribute to understanding human transcription initiation, to understanding human transcription regulation, and to design and synthesis of low-molecular-weight inhibitors of protein-DNA interactions in human transcription initiation, for application in selective regulation of transcription initiation in vitro and in vivo, and possibly for application in selective antiviral and anticancer therapy.