title: Operator-bound GalR dimers close DNA loops by direct interaction: tetramerization and inducer binding.
abstract: The assembly of the Gal repressosome, a higher order nucleoprotein complex that represses transcription of the gal operon in Escherichia coli, involves the formation of a DNA loop encompassing the promoter segment. GalR dimers bound to two spatially separated operators, O(E) and O(I), specifically interact with the histone-like protein HU and close the loop in supercoiled DNA. We isolated and characterized a GalR mutant containing an amino acid substitution (R282L) that can repress transcription in the absence of HU and supercoiled DNA both in vivo and in vitro. Repression involves the same DNA looping; deletion of either O(E) or O(I) makes the mutant GalR ineffective in repression. This and other results suggest that the R282L substitution increases the normal affinity between two DNA-bound GalR dimers, allowing looping. We conclude that GalR dimers interact directly and do not use HU as an adaptor in loop closure; HU and DNA supercoiling act in concert to stabilize the GalR tetramer. The stronger GalR-GalR interaction also made the gal transcription non-inducible, suggesting that the inducer binding acts by modulating tetramerization.
authors.affiliation: Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute/ National Institutes of Health, Bethesda, MD 20892-4264, USA.
authors.forename: Szabolcs
authors.initials: S
authors.lastname: Semsey
authors.forename: Mark
authors.initials: M
authors.lastname: Geanacopoulos
authors.forename: Dale E A
authors.initials: DE
authors.lastname: Lewis
authors.forename: Sankar
authors.initials: S
authors.lastname: Adhya
mesh_terms: D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D009154:Mutation; D009690:Nucleic Acid Conformation; D009875:Operator Regions, Genetic; D011485:Protein Binding; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C050993:Galactose repressor proteins; D012097:Repressor Proteins