Localization and Tracking of Single RNA Polymerase Molecules in Live E. Coli

Abstract

Superresolution fluorescence microscopy locates and tracks the diffusive motion of single copies of RNA polymerase (RNAP) in live E. coli. On a timescale of 0.1-1.0 s, most RNAP copies separate remarkably cleanly into two states with comparable populations. The “slow” RNAPs move indistinguishably from DNA loci. We assign them to specifically bound copies that are initiating transcription, elongating, pausing, or awaiting termination. The “fast” RNAP copies act as a homogeneous population with Dfast = 0.2 μm2-s-1. These are assigned as a rapidly exchanging mixture of non-specifically bound copies and copies undergoing three-dimensional diffusion within the nucleoids. In longer trajectories of 7-s duration, we directly observe transitions between the slow and fast states, corroborating the assignments. In rapid growth conditions, for which transcription of stable RNA predominates over transcription of mRNA, the slow, transcribing RNAP copies preferentially locate at the periphery of the nucleoids, as do rrnG and RNAP “transcription foci”. The data provide strong evidence for transcription occurring at or very near the cytoplasmic membrane, supporting the transertion hypothesis of co-transcriptional translation of membrane proteins. We compare the partitioning of RNAP states inferred from single-particle tracking with different partitioning models from the literature.