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

Abstract

By labeling the ␤ 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-m 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 ͳD RNAP ʹ of 0.22 ؎ 0.16 m 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 .In prokaryotes such as Escherichia coli, several thousand copies of a single RNA polymerase (RNAP) are responsible for all transcription. Previous work has used fluorescence microscopy to study the spatial distribution of green fluorescent protein (GFP)-labeled RNAP in both E. coli (4) and Bacillus subtilis (15) cells that were chemically fixed following growth in rich medium. In both species, narrow regions of high local GFP-RNAP concentration are observed atop a broader background concentration. Such "RNAP foci" are thought to be transcription foci, i.e., localized regions of intensive transcription, including rRNA. Accordingly, the narrow features were not observed for growth in minimal medium or following treatments designed to mimic nutrient starvation (4). The underlying cause of such spatial organization in prokaryotes is not clear (17). Quantifying the number, intensity, and spatial extent of RNAP foci in live cells is difficult due to the small size of the E. coli nucleoid, the presence of a background RNAP distribution, the dependence of the features on growth conditions, and the dynamic nature of the spatial distributions.Here we present a detailed quantitative study of the spatial distribution and movement of RNAP in live bacterial cells, specifically a K-12 strain of E. coli. The spatially narrow, immobile RNAP features that we observe appear qualitatively similar to the foci in fixed cells reported earlier. To capture the underlying dynamics, we imaged RNAP with a 40-ms time resolution for 0.8-s intervals. Each cell exhibits two or three narrow RNAP foci with a full width at half-maximum intensity (FWHM) of about 500 nm, significantly broader than the diffraction limit. These narrow features account for 20 to 30% of the total intensity and maintain their positions to within 60 nm over 1 s, during which time the broader background RNAP distribution fl...

Live-cell superresolution microscopy reveals the organization of RNA polymerase in the bacterial nucleoid

“…This analysis showed that ∼48% of RNAPs were bound and ∼52% were mobile (Fig. 1B, Inset); this result is in agreement with previous estimates from both fluorescence recovery after photobleaching (26) and single-molecule tracking (23). We then determined a D* threshold (0.16 μm 2 /s) which preserves the bound-to-mobile ratio, and allows sorting of individual trajectories as corresponding to bound or mobile RNAP molecules (SI Materials and Methods).…”

Direct interactions between the coiled-coil tip of DksA and the trigger loop of RNA polymerase mediate transcriptional regulation

“…To determine whether the DksA cross-link was to b or b9, we repeated the reaction with each of the six DksA-Bpa proteins and RNAP containing a b9 subunit with a C-terminal GFP fusion adding ;27 kDa to b9 (Bratton et al 2011). In all six cases, ;90%-95% of the radiolabeled cross-linked species migrated slightly slower than with wild-type RNAP to a position consistent with the molecular weight (MW) of a complex containing b9-GFP (155 kDa plus 27 kDa) cross-linked to DksA (18 kDa) (Fig.…”

Biochemical reactions in crowded environments: revisiting the effects of volume exclusion with simulations

“…However, to address the effect of crowding on the initiation of transcription, we first need to estimate whether this reaction is diffusion-limited. For a large protein complex such as RNA polymerase, the cytoplasmic diffusion coefficient is approximately 1 µm 2 /s [28,48], which is reduced due to non-specific binding to approximately 0.2 µm 2 /s [48,49]. Thus, the diffusion-limited binding rate to a promoter can be estimated to be about 0.1 µM −1 s −1 .…”