By studying the mechanism of Gal repressor (GalR) action in Escherichia coli, we have uncovered several new aspects about the mechanism of transcription and its regulation. (1)Repression by repressosome: Transcription from two overlapping galpromoters, P1 and P2, is repressed by GalR binding to bipartite operators, OE and OI, which flank the promoters. Concurrent repression of P1 and P2 also requires the bacterial histone-like protein, HU, acting as a cofactor. Footprinting experiments using HU chemically converted into a nuclease showed that HU binding is site specific and dependant upon GalR binding to OE and OI and supercoiled DNA. Repression requires the formation of a higher order nucleoprotein complex (repressosome) in which the two DNA-bound GalR interact with each other forming a DNA loop. By isolating GalR mutants, we have identified surface exposed residues both in GalR and HU which are involved in protein-protein interaction(s) in repressosome formation. (2) Repression by Trapping RNA polymerase: In the absence of DNA looping, GalR bound to the upstream operator, OE, acts as repressor of P1 and as activator of P2. GalR performs this dual role by making contacts with specific amino acids of the C-terminal domain of the alpha subunit of DNA- bound RNA polymerase. By genetic and biochemical analysis, we have defined the sites in alpha CTD that are contacted by GalR for repression and activation. By following 2, aminopurine fluorescence, we have assayed individual steps of transcription initiation at the P1 promoter and have shown that GalR inhibits an intermediate step (isomerization) in the initiation process. (3) Transcription Elongation: Our results also suggest that there may be a reading phase when RNA polymerase transcribes DNA; we have found that recognition of a transcription barrier is related to its distance from the promoter in a periodic fashion. - anisotropy, atomic force microscope, DNA loop, fluorescence, HU, inducer, repressor, RNA polymerase, Transcription, - Neither Human Subjects nor Human Tissues
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