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

Subhas C. Bera, Pim P.B. America, Santeri Maatsola, Mona Seifert, Eugeniu Ostrofet, Jelmer Cnossen, Monika Spermann, Martin Depken, David Dulin*, More Authors

*Corresponding author for this work

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Abstract

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 σ 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 σ and the RNAP β subunit. The temperature dependence provides the energy scale of open-complex formation and further supports the existence of additional intermediates.

Original languageEnglish
Pages (from-to)7511-7528
JournalNucleic Acids Research
Volume50
Issue number13
DOIs
Publication statusPublished - 2022

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