The principal objectives of the proposed research are (a) to investigate the structure and dynamics of the DNA-dependent RNA polymerase, and related regulatory proteins, (b) to resolve the complex processes of RNA synthesis into their elementary steps, and (c) to elucidate the control mechanism of gene transcription. We propose to approach these objectives by various physicochemical methods, in particular, fast kinetics, nanosecond fluorescence spectroscopy and various cross-linking techniques. In this way, both structural and dynamic information will be yielded. Studies will be carried out in both prokaryotic and eukaryotic systems for the gene transcription in Escherichia coli and the Xenopus 5S RNA synthesis. The problems to be investigated in the Escherichia coli system include: (a) the structural and functional relationship among the multiple binding sites and subunits of RNA polymerase, (b) the molecular topography of RNA polymerase-promoter recognition and the kinetic mechanisms of promoter search, (c) the elementary steps involved in RNA chain initiation and elongation, and (e) the syncatalytic mapping and the translocation of the enzyme during the course of RNA synthesis. In the Xenopus system, we will study (a) the molecular mechanism involved in the factor A-5S RNA gene interaction that leads to specific transcription initiation, (b) the ability of 5S RNA to compete with its gene for factor A in an autoregulatory manner, and (c) the purification and characterization of the transcription complexes for the 5S RNA synthesis in vitro. The ultimate goal of this study is to understand the mechanism and regulation of gene expression in molecular detail.
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