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, KB) and isomerization of the closed complex to an active, stable, open complex (described by a forward rate constant, kf). λ cI protein activates the PRMpromoter by specifically increasing kf. A positive control mutant, cI-pc2, is defective for activation because it fails to raise kf. An Arg to His change in the σ70subunit of RNA polymerase was previously obtained as an allele-specific suppressor of cI-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 alter KBor kfin 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 increasing kf. An unexpected and novel finding is that the wild-type activator stimulates the mutant polymerase, but not wild-type polymerase, by increasing KB.