Intrinsic transcription termination is a process whereby ternary elongation complex (TEC) dissociates into RNA transcript, DNA template, and RNA polymerase in response to specific signals encoded in the DNA. In bacteria, such signals are evolutionarily conserved and appear in RNA as a stable secondary structure (a hairpin) followed by a run of uridine residues at the 3' terminus. Intrinsic termination is an integral part of the transcription cycle and has a crucial role in the regulation of gene expression at the level of RNA synthesis. The long term objective of the proposed work is to provide a comprehensive structural and mechanistic description of the intrinsic transcription termination by Escherichia coli RNA polymerase and the detailed mechanisms of its regulation by elongation factors.
The specific aims of the proposal are: 1) Analysis of protein-RNA and protein-DNA interactions in the termination complex. Experiments are proposed to monitor the rearrangement of protein-RNA and protein-DNA contacts in the TEC when it reaches the termination site. 2) Analysis of protein-RNA and protein-DNA interactions in the antitermination complex. Experiments are proposed to determine the effects of E. coli elongation factor NusA and phage lambda antitermination factor N on the protein-nucleic acids interactions in the TEC during termination. 3) Determination of the low resolution structure of the elongation and termination complexes. Experiments re proposed to determine 15 -20 angstrom resolution structures if the TEC which either carries a single stranded RNA or the termination hairpin. Differences between two structures will allow a detection of the major conformational changes associated with termination.
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