Abstract
DNA transaction reactions require formation of nucleoprotein complexes that involve multifaceted DNA-protein and protein-protein interactions. Genetic and biochemical studies suggested that the higher order Gal repressosome structure, which governs the transcription of two tandem galpromoters in Escherichia coli, involves sequence-specific binding of GalR repressor dimers to two operators, O(E) and O(I), located 113 bp apart, binding of GalR to the sequence-nonspecific DNA binding protein HU, interaction of HU with an architecturally critical DNA site between the two operators, and interaction between two DNA-bound GalR dimers generating a loop of the intervening DNA segment. In this paper, we demonstrate and determine the thermodynamic parameters of several of these interactions, GalR dimer-O(E), GalR tetramerization, HU-GalR, and HU-GalR-O(E) interactions, by analytical ultracentrifugation, fluorescence anisotropy, and fluorescence resonance energy transfer. The physiological significance of several of these interactions was confirmed by the finding that a mutant HU, which is unable to form the repressosome in vivo and in vitro, failed to show the HU-GalR interaction. The results help to construct a pathway of Gal repressosome assembly.
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