An RNA polymerase mutant deficient in DNA melting facilitates study of activation mechanism: application to an artificial activator of transcription.

Transcription initiation is a major target for the regulation of gene expression in all organisms. Transcription activators can stimulate different steps in the initiation process including the initial binding of RNA polymerase (RNAP) to the promoter and a subsequent promoter-melting step. Typically, kinetic assays are required to determine whether an activator exerts its effect on the initial binding of RNAP or on the promoter-melting step. Here we take advantage of a mutant Escherichia coli RNAP that is deficient in promoter melting to assess the ability of an activator to stabilize the initial binding of RNAP to the promoter. For the well-characterized activator CRP, we show that this RNAP mutant can be used to distinguish between effects on initial binding and promoter melting; these results provide an independent confirmation of the results of kinetic analysis. We then employ the melting-deficient RNAP mutant to demonstrate an effect of an artificial activator of transcription on the initial binding of RNAP. Our findings demonstrate that a melting-deficient RNAP mutant can be used to trap a normally unstable intermediate in transcription initiation, thus providing a novel tool for probing activation mechanism.