Mechanism of activation of transcription initiation from the λPRM promoter

Abstract

The mechanism of activation of the bacteriophage λP RM promoter by the product of the λcI gene, the λ repressor, was studied in vitro. We found that repressor increased the rate of RNA polymerase open complex formation at P RM; the final extent of promoter occupancy and the catalytic properties of the open complexes were unaffected by the presence of repressor. Separate quantitation of the two steps of open complex formation showed that repressor specifically enhanced the rate of the second step, the isomerization of the closed complex to the transcriptionally active open complex. The first step, the initial binding of the RNA polymerase to the promoter to form the closed complex, was not significantly affected by repressor. We also studied the activation of a −35 region mutant of P RM, prmup-1, and found that the mechanism of activation was analogous to that of the wild-type promoter, even though the prmup-1 promoter initiates more frequently than P RM in the absence of repressor. Finally, we present evidence for a stabilizing interaction between RNA polymerase at P RM and repressor at O R2 and demonstrate that repressor is required only for the formation, but not for the maintenance, of the open complex. The comparison of the in vitro initiation properties of the wild-type and prmup-1 promoters revealed an effect of the mutation on both steps of open complex formation. This result is analogous to the observation that a −35 mutation in P R, x3, decreased the favorability of both steps (Hawley & McClure, 1980). We propose a model in which the mutation directly affects only one step, the initial binding, while the observed effect on the isomerization rate is indirect. We present a detailed discussion of this model in the Appendix.