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. The gal repression system provides a model for studying how a """"""""condensed"""""""" DNA becomes available for transcription. 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. 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. 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 a subunit of RNA polymerase, we proposed that the inducer binding to the OE-GalR allosterically relieves the inhibitory effect without dissociating GalR from OE.
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