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

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.

High spatiotemporal resolution data from a custom magnetic tweezers instrument.

Gene expression is achieved by enzymes as RNA polymerases that translocate along nucleic acids with steps as small as a single base pair, i.e., 0.34 nm for DNA. Deciphering the complex biochemical pathway that describes the activity of such enzymes requires an exquisite spatiotemporal resolution. Magnetic tweezers are a powerful single molecule force spectroscopy technique that uses a camera-based detection to enable the simultaneous observation of hundreds of nucleic acid tethered magnetic beads at a constant force with subnanometer resolution [1,2]. High spatiotemporal resolution magnetic tweezers have recently been reported [3-5]. We present data acquired using a bespoke magnetic tweezers instrument that is able to perform either in high throughput or at high resolution. The data reports on the best achievable resolution for surface-attached polystyrene beads and DNA tethered magnetic beads, and highlights the influence of mechanical stability for such assay. We also present data where we are able to detect 0.3 nm steps along the z-axis using DNA tethered magnetic beads. Because the data presented here are in agreement with the best resolution obtained with magnetic tweezers, they provide a useful benchmark comparison for setup adjustment and optimization.