Gal repressor-operator-HU ternary complex: pathway of repressosome formation.
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.
Roy
Siddhartha
S
Department of Biophysics, Bose Institute, Calcutta 700019, India.
Dimitriadis
Emilios K
EK
Kar
Sudeshna
S
Geanacopoulos
Mark
M
Lewis
Marc S
MS
Adhya
Sankar
S
D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D009875:Operator Regions, Genetic; D012097:Repressor Proteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014461:Ultracentrifugation
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C050993:Galactose repressor proteins; D009838:Oligodeoxyribonucleotides; D012097:Repressor Proteins; C116822:hns protein, E coli