Changing the mechanism of transcriptional activation by phage λ repressor

The first steps of transcription initiation include binding of RNA polymerase to a promoter to form an inactive, unstable, closed complex (described by an equilibrium constant,K B ) and isomerization of the closed complex to an active, stable, open complex (described by a forward rate constant,k f ). λ cI protein activates theP RM promoter by specifically increasingk f . A positive control mutant, cI-pc2, is defective for activation because it fails to raisek f . An Arg to His change in the σ70 subunit of RNA polymerase was previously obtained as an allele-specific suppressor ofcI-pc2 . To elucidate how the mutant polymerase restores the activation function of the mutant activator, abortive initiation assays were performed, using purified cI proteins and RNA polymerase holoenzymes. The change in σ does not significantly alterK B ork f in the absence of cI protein. As expected, cI-pc2 activates the mutant polymerase in the same way that wild-type cI activates the wild-type polymerase, by increasingk f . An unexpected and novel finding is that the wild-type activator stimulates the mutant polymerase, but not wild-type polymerase, by increasingK B .