Mechanistic Differences in Promoter DNA Melting by Thermus aquaticus and Escherichia coli RNA Polymerases

Formation of strand-separated, functional complexes at promoters was compared for RNA polymerases from the mesophile Escherichia coli and the thermophile Thermus aquaticus. The RNA polymerases contained sigma factors that were wild type or bearing homologous alanine substitutions for two aromatic amino acids involved in DNA melting. Substitutions in the σA subunit of T. aquaticus RNA polymerase impair promoter DNA melting equally at temperatures from 25 to 75 °C. However, homologous substitutions in σ70 render E. coli RNA polymerase progressively more melting-defective as the temperature is reduced below 37 °C. The effects of the mutations on the mechanism of promoter DNA melting were investigated by studying the interaction of wild type and mutant RNA polymerases with “partial promoters” mimicking promoter DNA where the nucleation of DNA melting had taken place. Because T. aquaticus and E. coli RNA polymerases bound these templates similarly, it was concluded that the different effects of the mutations on the two polymerases are exerted at a step preceding nucleation of DNA melting. A model is presented for how this mechanistic difference between the two RNA polymerase could explain our observations.