Abstract
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 sigmaA subunit of T. aquaticus RNA polymerase impair promoter DNA melting equally at temperatures from 25 to 75 degrees C. However, homologous substitutions in sigma70 render E. coli RNA polymerase progressively more melting-defective as the temperature is reduced below 37 degrees 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.
Highlights
Transcription in bacteria is catalyzed by DNA-dependent RNA polymerase (RNAP),1 a key enzyme for gene expression and its regulation [1, 2]
Template Design—To compare the temperature dependence for open complex formation by the Thermus aquaticus (Taq) and Escherichia coli (Eco) RNAP under the conditions employed in our experiments, the same Duplex promoter DNA template (Fig. 1) was used, taking advantage of the fact that the primary sigma factors of the two RNAP have similar promoter specificities
Our results suggest that the Duplex sequence is a better promoter for Eco RNAP at 37 °C than for Taq RNAP at 65 °C (Fig. 2)
Summary
Transcription in bacteria is catalyzed by DNA-dependent RNA polymerase (RNAP), a key enzyme for gene expression and its regulation [1, 2]. Studies with Escherichia coli (Eco) RNAP have indicated that upon RNAP binding to promoter DNA, an unstable closed complex (RPc) is formed, followed by at least two additional intermediates (I1 and I2) before the initiation-competent, stable open complex (RPo) is formed [9, 10], in which a 14-bp region of the promoter DNA, including the start site of transcription [2], has been melted. In this work we have compared Taq and Eco RNAP-containing sigma factors that were both wt or bearing homologous amino acid substitutions in region 2.3, with respect to their ability to interact with promoters and with model templates designed to emulate the state of promoter DNA in various intermediates of scheme 1. The difference would lie in the RPc to I1 conversion, which would be affected by both temperature and the mutations for Eco RNAP but only one or the other for Taq RNAP
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