{Reference Type}: Journal Article
{Title}: Quantitative parameters of bacterial RNA polymerase open-complex formation, stabilization and disruption on a consensus promoter.
{Author}: Bera SC;America PPB;Maatsola S;Seifert M;Ostrofet E;Cnossen J;Spermann M;Papini FS;Depken M;Malinen AM;Dulin D;
{Journal}: Nucleic Acids Res
{Volume}: 50
{Issue}: 13
{Year}: 07 2022 22
{Factor}: 19.16
{DOI}: 10.1093/nar/gkac560
{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 $\sigma$ 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.