Quantitative parameters of bacterial RNA polymerase open-complex formation, stabilization and disruption on a consensus promoter

• Published in: Nucleic acids research 2022-07, Vol.50 (13), p.7511-7528
• Main Authors: Bera, Subhas C, America, Pim P B, Maatsola, Santeri, Seifert, Mona, Ostrofet, Eugeniu, Cnossen, Jelmer, Spermann, Monika, Papini, Flávia S, Depken, Martin, Malinen, Anssi M, Dulin, David
• Format: Article
• Language: English
• Subjects: Nucleic Acid Enzymes
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkac560

Transcription initiation is the first step in gene expression, and is therefore strongly regulated in all domains of life. The RNA polymerase (RNAP) first associates with the initiation factor $\sigma$ to form a holoenzyme, which binds, bends and opens the promoter in a succession of reversible states. These states are critical for transcription regulation, but remain poorly understood. Here, we addressed the mechanism of open complex formation by monitoring its assembly/disassembly kinetics on individual consensus lacUV5 promoters using high-throughput single-molecule magnetic tweezers. We probed the key protein–DNA interactions governing the open-complex formation and dissociation pathway by modulating the dynamics at different concentrations of monovalent salts and varying temperatures. Consistent with ensemble studies, we observed that RNAP-promoter open (RPO) complex is a stable, slowly reversible state that is preceded by a kinetically significant open intermediate (RPI), from which the holoenzyme dissociates. A strong anion concentration and type dependence indicates that the RPO stabilization may involve sequence-independent interactions between the DNA and the holoenzyme, driven by a non-Coulombic effect consistent with the non-template DNA strand interacting with $\sigma$ and the RNAP $\beta$ subunit. The temperature dependence provides the energy scale of open-complex formation and further supports the existence of additional intermediates.