RESUMO
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.
Assuntos
RNA Polimerases Dirigidas por DNA , Escherichia coli , Regiões Promotoras Genéticas , Bactérias/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Holoenzimas/genética , Holoenzimas/metabolismo , RNA Bacteriano , Fator sigma/metabolismo , Transcrição GênicaRESUMO
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., 0.34â¯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 0.3â¯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.