KMnO4 as a probe for lac promoter DNA melting and mechanism in vivo

Abstract

The reagent potassium permanganate is used to probe lac transcription complexes by primer extension-probing analysis. A series of strongly hyperreactive bands, corresponding to the known melted region, is observed when open complexes are formed in vitro. A nearly identical pattern occurs in vivo, the signal intensity of which increases when open complexes are trapped with rifampicin. Quantitative comparison of the signal intensity obtained under steady-state conditions with that obtained in the presence of rifampicin indicates that transcription from each of three lac promoter variants is limited in vivo principally by the slow rate of open complex formation. The slow-start lac L8:UV5 promoter is also limited somewhat by slow RNA chain initiation. Slightly different patterns of KMnO4 reactivity at each promoter variant in the absence of RNA polymerase in vitro suggest that DNA sequence dictates the ultimate pattern of melting, with the polymerase acting principally to stabilize the melted state specified by the DNA sequence.