Transcription constitutes the central point of control for gene expression in the cell. The eukaryotic Pol II enzyme must transcribe in the presence of the physical barriers present in nucleosomal DNA and both this enzyme and its prokaryotic counterpart must transcribe torsionally constrained DNA segments that require the enzymes to translocate against increasing torques. Moreover, during transcription elongation these two highly structurally homologous enzymes perform a mechanochemical cycle in which a nucleotide condensation reaction is coupled to the mechanical tasks of movement, force, and torque generation. In this application we propose to use a combination of imaging and single molecule manipulation approaches to: a) Investigate the dynamics of transcription through nucleosomal DNA by yeast Pol II. b) Determine the effect of elongation factors on nucleosomal transcription. c) Investigate the effect of histone modification on the dynamics of nucleosomal transcription. d) Investigate the effect of remodeling factors on the dynamics of Pol II through nucleosomal DNA. We also propose to continue our previous studies of the prokaryotic E. coli RNA polmerase enzyme to investigate: a) Its generation of torque during transcription of torsionally constrained DNA, and b) The details of its mechanochemical cycle at high spatial resolution.
Transcription constitutes the central point of control for regulation of gene expression. Disruption of this process is associated with congenital heart disease, oncogenesis, and a large variety of developmental pathologies. The insights that our experiments provide on transcription will support the first steps in the development of future therapies.
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