PMID:15807530
| Citation |
Roy, S, Dimitriadis, EK, Kar, S, Geanacopoulos, M, Lewis, MS and Adhya, S (2005) Gal repressor-operator-HU ternary complex: pathway of repressosome formation. Biochemistry 44:5373-80 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1021/bi047720t |
| Keywords |
Base Sequence; DNA-Binding Proteins/metabolism; Escherichia coli Proteins/metabolism; Oligodeoxyribonucleotides; Operator Regions, Genetic; Repressor Proteins/metabolism; Spectrometry, Fluorescence; Ultracentrifugation |
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