Partitioning of RNA polymerase activity in live Escherichia coli from analysis of single-molecule diffusive trajectories.

Superresolution fluorescence microscopy is used to locate single copies of RNA polymerase (RNAP) in live Escherichia coli and track their diffusive motion. On a timescale of 0.1-1 s, most copies separate remarkably cleanly into two diffusive states. The "slow" RNAPs, which move indistinguishably from DNA loci, are assigned to specifically bound copies (with fractional population ftrxn) that are initiating transcription, elongating, pausing, or awaiting termination. The "mixed-state" RNAP copies, with effective diffusion constant Dmixed = 0.21 μm(2) s(-1), are assigned as a rapidly exchanging mixture of nonspecifically bound copies (fns) and copies undergoing free, three-dimensional diffusion within the nucleoids (ffree). Longer trajectories of 7-s duration reveal transitions between the slow and mixed states, corroborating the assignments. Short trajectories of 20-ms duration enable direct observation of the freely diffusing RNAP copies, yielding Dfree = 0.7 μm(2) s(-1). Analysis of single-particle trajectories provides quantitative estimates of the partitioning of RNAP into different states of activity: ftrxn = 0.54 ± 0.07, fns = 0.28 ± 0.05, ffree = 0.12 ± 0.03, and fnb = 0.06 ± 0.05 (fraction unable to bind to DNA on a 1-s timescale). These fractions disagree with earlier estimates.