%0 Journal Article
%T Quantitative parameters of bacterial RNA polymerase open-complex formation, stabilization and disruption on a consensus promoter.
%A Bera SC
%A America PPB
%A Maatsola S
%A Seifert M
%A Ostrofet E
%A Cnossen J
%A Spermann M
%A Papini FS
%A Depken M
%A Malinen AM
%A Dulin D
%J Nucleic Acids Res
%V 50
%N 13
%D 07 2022 22
%M 35819191
%F 19.16
%R 10.1093/nar/gkac560
%X 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.