In vitro transcription close to the melting point of DNA: Analysis of Thermotoga maritima RNA polymerase - promoter complexes at 75°C using chemical probes

The interaction of DNA dependent RNA polymerase of the extreme thermophile bacteria Thermotoga maritima with a promoter bearing DNA fragment was investigated in the temperature range from 20 to 85°C. We show that the T.maritima RNA polymerase recognizes and utilizes the Escherichia coli T7 A1 promoter with an efficiency similar to that of the E.coli polymerase. We have investigated the interaction of both polymerases with the same promoter over a wide range of temperatures using hydroxyl radical foot-printing and osmium tetroxide probing. This study revealed that the T.maritima polymerase goes through a series of isomerisation events very similarto the E.coli polymerase, i.e. the closed, intermediate and open complexes, but the transitions themselves occur at radically different temperatures. This indicates that conformational changes in the DNA that accompany initiation of transcription such as promoter melting are determined by the polymerase rather than the DNA sequence.