GaIR represses gaIP1 by inhibiting the rate-determining open complex formation through RNA polymerase contact: A GaIR negative control mutant

Abstract

Ga1R represses the galP1 promoter by a DNA looping-independent mechanism. Equilibrium binding of Ga1R 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 Ga1R, 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, Ga1R inhibited the slower of the two-step base-pair distortion process. We isolated a negative control Ga1R 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 Ga1R establishes contact with alpha-CTD at the last resolved isomerization. intermediate, forming a kinetic trap.