An Escherichia coli RNA polymerase tight-binding site on T7 DNA is a weak promoter subject to substrate inhibition

Abstract

A specific Escherichia coli RNA polymerase tight-binding (TB) site on bacteriophage T7 has been located at 32,988 base pairs from the left end of T7. This site is referred to as the T7 F promoter since it is fully active in vitro. Kinetics of association and dissociation have been measured by use of the abortive initiation assay and runoff transcription. The association constant, ka approximately 9 X 10(5) M-1 s-1, is of the same magnitude as ka for the T7 minor promoters. In competitive titration assays, the F promoter was found to be slightly weaker than the minor T7 E promoter at low RNA polymerase concentrations and, as expected, much weaker than the major T7 A3 promoter. An unusual RNA polymerase mediated inhibition of both the association rate and the transcriptional activity was observed at moderately high concentrations of polymerase. A mechanistic model analogous to enzyme substrate inhibition is presented.