CytR/cAMP-CRP nucleoprotein formation in E. coli: the CytR repressor binds its operator as a stable dimer in a ternary complex with cAMP-CRP.
The CytR repressor protein relies on protein-protein interactions to the cAMP-CRP complex to bind its operators with sufficiently high affinity to repress transcription. Here, the quaternary structure of CytR and the mechanism underlying the cooperative binding of CytR and cAMP-CRP have been analyzed. Using a modified Ferguson analysis in which protein-DNA complexes are separated in a non-denaturing gel system, we show that CytR binds its operators as a dimer alone as well as in a ternary complex with cAMP-CRP. Analyses of DNA binding of CytR at low protein concentrations indicate that CytR is a dimer in solution at physiological concentrations. Moreover, the CytR inducer cytidine was found not to have any effect on the oligomerization of free CytR or DNA bound CytR. Thus, these data rule out the possibility that the cooperative DNA binding of CytR and cAMP-CRP involves induced dimerization of CytR, and they suggest that cytidine interrupts the cooperative binding of CytR and cAMP-CRP solely by perturbing the protein-protein interactions between the two proteins.
Kristensen
H H
HH
Department of Molecular Biology, University of Odense, Denmark.
Valentin-Hansen
P
P
Søgaard-Andersen
L
L
D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D002352:Carrier Proteins; D016384:Consensus Sequence; D000242:Cyclic AMP / Q000378:metabolism; D002373:Cyclic AMP Receptor Protein / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008956:Models, Chemical; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D009875:Operator Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; C048425:CytR protein, E coli; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D012097:Repressor Proteins; D000242:Cyclic AMP