Studies of contacts between T7 RNA polymerase and its promoter reveal features in common with multisubunit RNA polymerases

We have used UV-laser mediated cross-linking, DNase I footprinting and KMnO 4 reactivity to probe the interaction between T7 RNA polymerase (RNAP) and a consensus promoter during the early stages of transcription. In a binary complex formed in the absence of substrate on a supercoiled plasmid, direct contacts were observed on the template (T) strand at positions -17, -5, an d +3 and on the nontemplate (NT) strand at position -8. These contacts lie within the DNase I cleavage footprint from positions -21 to +11 on the T st rand and from positions -17 to +16 on the NT strand and straddle sites of e nhanced reactivity of thymines to KMnO4 at position -3 on the T strand and position -2 on the NT strand. Use of supercoiled plasmid templates has allo wed the mapping of contacts in the initiation region of the promoter in the binary complex for the first time. Upon addition of GTP, T7 RNAP enters a reiterative mode of synthesis, producing a ladder of poly(G) products. Unde r these conditions the downstream contact on the T strand switched from pos ition +3 to +4 and +5 while the contact at position -17 was maintained. Und er conditions in which the synthesis of transcription products is limited t o 6-7 nucleotides, only the contact at position -17 on the T strand was pre served. A comparison of these results with the interaction of Escherichia c all RNA polymerase at the Inc promoter reveals strong similarities in the m anner in which these polymerases recognize their promoters.