GalR represses galP1 by inhibiting the rate-determining open complex formation through RNA polymerase contact: a GalR negative control mutant.
GalR represses the galP1 promoter by a DNA looping-independent mechanism. Equilibrium binding of GalR and RNA polymerase to DNA, and real-time kinetics of base-pair distortion (isomerization) showed that the equilibrium dissociation constant of RNA polymerase-P1 closed complexes is largely unaffected in the presence of saturating GalR, indicating that mutual antagonism (steric hindrance) of the regulator and the RNA polymerase does not occur at this promoter. In fluorescence kinetics with 2-AP labeled P1 DNA, GalR inhibited the slower of the two-step base-pair distortion process. We isolated a negative control GalR mutant, S29R, which while bound to the operator DNA was incapable of repression of P1. Based on these results and previous demonstration that repression requires the C-terminal domain of the alpha subunit (alpha-CTD) of RNA polymerase, we propose that GalR establishes contact with alpha-CTD at the last resolved isomerization intermediate, forming a kinetic trap.
Roy
Siddhartha
S
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Semsey
Szabolcs
S
Liu
Mofang
M
Gussin
Gary N
GN
Adhya
Sankar
S
D001483:Base Sequence; D004247:DNA; D018983:DNA Footprinting; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins; D005454:Fluorescence Polarization; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic
D029968:Escherichia coli Proteins; C050993:Galactose repressor proteins; D012097:Repressor Proteins; D004247:DNA; D012321:DNA-Directed RNA Polymerases