RNA polymerase-promoter interactions determining different stability of the Escherichia coli and Thermus aquaticus transcription initiation complexes.

Vladimir Mekler, Leonid Minakhin, Konstantin Kuznedelov, Damir Mukhamedyarov, Konstantin Severinov

Nucleic acids research 2012 Dec

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Transcription initiation complexes formed by bacterial RNA polymerases (RNAPs) exhibit dramatic species-specific differences in stability, leading to different strategies of transcription regulation. The molecular basis for this diversity is unclear. Promoter complexes formed by RNAP from Thermus aquaticus (Taq) are considerably less stable than Escherichia coli RNAP promoter complexes, particularly at temperatures below 37°C. Here, we used a fluorometric RNAP molecular beacon assay to discern partial RNAP-promoter interactions. We quantitatively compared the strength of E. coli and Taq RNAPs partial interactions with the -10, -35 and UP promoter elements; the TG motif of the extended -10 element; the discriminator and the downstream duplex promoter segments. We found that compared with Taq RNAP, E. coli RNAP has much higher affinity only to the UP element and the downstream promoter duplex. This result indicates that the difference in stability between E. coli and Taq promoter complexes is mainly determined by the differential strength of core RNAP-DNA contacts. We suggest that the relative weakness of Taq RNAP interactions with DNA downstream of the transcription start point is the major reason of low stability and temperature sensitivity of promoter complexes formed by this enzyme.

Citation

Vladimir Mekler, Leonid Minakhin, Konstantin Kuznedelov, Damir Mukhamedyarov, Konstantin Severinov. RNA polymerase-promoter interactions determining different stability of the Escherichia coli and Thermus aquaticus transcription initiation complexes. Nucleic acids research. 2012 Dec;40(22):11352-62