E.Coli Rna Polymerase Activity Under Crowding

Biological reactions in the cellular environment differ physicochemically from those performed in dilute buffer solutions due to the high viscosity and crowding effects associated with the high density of macromolecules in the cell. Earlier work has shown that in vivo bacterial transcription is affected by the cellular environment, involving slower diffusion of various components in the cellular milieu (due to high viscosity), increase in their local concentrations, and modulation of their binding affinities. However, since most studies are focused on multiple cycles of either transcription or association of DNA and RNA Polymerase (RNAP) to make RNAP open complex (RPo), the effect of the crowded environment on a single transcription run is still poorly understood. Here, we have developed and applied a novel transcription quenched-kinetics assay using single-molecule detection to investigate the size/concentration effects of various osmolytes and macromolecular crowding agents, which mimic the crowded cellular environment, on actively-transcribing RNAP. Our results demonstrate an expected slowdown of transcription kinetics due to increased viscosity, and unexpected enhancement in transcription activity by larger crowding agents at the same viscosity. These findings suggest that crowding agents affect the transcription reaction after RPo formation through volume exclusion.