GalR-dependent Site-specific Binding of HU for gal Repression: Transcription from two overlapping gal promoters is repressed by Gal repressor binding to bipartite gal operators, OE and OI, which flank the promoters. Concurrent repression of the gal promoters also requires the bacterial histone-like protein HU which acts as a co-factor. Footprinting experiments using iron EDTA-coupled HU show that HU binding to gal DNA is site-specific and specifically dependent upon binding of GalR to both OE and OI. HU, in concert with GalR, forms a specific nucleoprotein higher order complex containing a DNA loop. This way, HU deforms the promoter to make the latter inactive for transcription initiation while remaining sensitive to inducer. We have isolated and characterized GalR mutants which bind to the operators normally but are defective in repression. These mutations define either GalR-GalR or GalR-HU contact sites in the nucleoprotein repression complex. The proposed protein-protein contact sites are also being established by fluorescence spectroscopy. Atomic Force Microscopic Demonstration of DNA Looping: Applying atomic force microscope imaging to visualize gal DNA complexes with proteins, we observed GalR mediated DNA looping in which HU plays an obligatory role by helping GalR tetramerization. Repression without DNA Looping: 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. The contact sites are being studied by fluorescence spectroscopy. Mechanism of Induction of P1: Thermodynamic and fluorescence anisotropy experiments suggest that the inducer, D-galactose, binding to the OE- GalR complex does not dissociate GalR from OE for transcription induction. Since GalR inhibits transcription by modulating the alpha subunit of RNA polymerase, we proposed that the inducer binding to the OE-bound GalR allosterically relieves the inhibitory effect without dissociating GalR from OE.

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