Spatial Distribution and Diffusive Motion of RNA Polymerase in Live Escherichia coli

Abstract

ABSTRACT By labeling the β′ subunit of RNA polymerase (RNAP), we used fluorescence microscopy to study the spatial distribution and diffusive motion of RNAP in live Escherichia coli cells for the first time. With a 40-ms time resolution, the spatial distribution exhibits two or three narrow peaks of 300- to 600-nm full width at half-maximum that maintain their positions within 60 nm over 1 s. The intensity in these features is 20 to 30% of the total. Fluorescence recovery after photobleaching (FRAP) measures the diffusive motion of RNAP on the 1-μm length scale. Averaged over many cells, 53% ± 19% of the RNAP molecules were mobile on the 3-s timescale, with a mean apparent diffusion constant 〈 D RNAP 〉 of 0.22 ± 0.16 μm 2 -s −1 . The remaining 47% were immobile even on the 30-s timescale. We interpret the immobile fraction as arising from RNAP specifically bound to DNA, either actively transcribing or not. The diffusive motion of the mobile fraction ( f mobile ) probably involves both one-dimensional sliding during nonspecific binding to DNA and three-dimensional hopping between DNA strands. There is significant cell-to-cell heterogeneity in both D RNAP and f mobile .