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

Thomas Meier, Peter Schickor, Andrew Wedel, Luciano Cellai, Hermann Heumann

Research output: Contribution to journalArticle

23 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)988-994
Number of pages7
JournalNucleic acids research
Volume23
Issue number6
DOIs
StatePublished - Mar 25 1995
Externally publishedYes

ASJC Scopus subject areas

  • Genetics

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