CAP interacts with RNA polymerase in solution in the absence of promoter DNA.
Protein-protein interactions between transcription activator proteins and RNA polymerase or basal transcription factors have been suggested to be important for transcription activation. Interactions between catabolite gene activator protein (CAP) and RNA polymerase have been proposed based on face-of-helix-dependent transcription activation by CAP and based on face-of-helix-dependent cooperative binding of CAP and RNA polymerase to promoter DNA. Mutants of CAP specifically defective in transcription activation have been isolated (mutants defective in transcription activation, but not defective in DNA binding and DNA bending). All such mutants contain amino-acid substitutions within a surface loop consisting of amino acids 152 to 166 of CAP. Here we use the thermodynamically rigorous technique of fluorescence polarization to show that CAP interacts with RNA polymerase in solution in the absence of promoter DNA (KD,app = 2.8 x 10(-7) M), whereas [Ala158]CAP, a mutant of CAP specifically defective in transcription activation, does not.
Heyduk
T
T
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77550.
Lee
J C
JC
Ebright
Y W
YW
Blatter
E E
EE
Zhou
Y
Y
Ebright
R H
RH
D001483:Base Sequence; D002373:Cyclic AMP Receptor Protein / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005454:Fluorescence Polarization; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012996:Solutions
D002373:Cyclic AMP Receptor Protein; D012996:Solutions; D004247:DNA; D012321:DNA-Directed RNA Polymerases