Activate Transcription Initiation Research Articles

The effect of a lambda repressor mutation on the activation of transcription initiation from the lambda PRM promoter

Wild-type λ repressor activates transcription from the λ P RM promoter by stimulating the rate-limiting isomerization step in the initiation reaction. The positive-control mutants of λ repressor retain the ability to bind operator DNA normally, but fail to activate transcription from the λ P RM promoter in vivo. We have characterized one of these mutants in vitro, and have determined the biochemical nature of the defect. We show that the mutant repressor was deficient in its ability to stimulate the isomerization step in transcription initiation. The initial binding of RNA polymerase to P RM was only slightly reduced by the mutant repressor. We also found that the mutant and wild-type repressors had similar affinities for all three binding sites in the rightward operator. These results provide support for the hypothesis that direct repressor-RNA polymerase interactions are important in the P RM activation mechanism.

Mechanism of activation of transcription initiation from the λPRM promoter

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.