Multiscale Spatial Organization of RNA Polymerase in Escherichia coli

Abstract

Nucleic acid synthesis is spatially organized in many organisms. In bacteria, however, the spatial distribution of transcription remains obscure, owing largely to the diffraction limit of conventional light microscopy (200-300 nm). Here, we use photoactivated localization microscopy to localize individual molecules of RNA polymerase (RNAP) in Escherichia coli with a spatial resolution of ∼40 nm. In cells growing rapidly in nutrient-rich media, we find that RNAP is organized in 2-8 bands. The band number scaled directly with cell size (and so with the chromosome number), and bands often contained clusters of >70 tightly packed RNAPs (possibly engaged on one long ribosomal RNA operon of 6000 bp) and clusters of such clusters (perhaps reflecting a structure like the eukaryotic nucleolus where many different ribosomal RNA operons are transcribed). In nutrient-poor media, RNAPs were located in only 1-2 bands; within these bands, a disproportionate number of RNAPs were found in clusters containing ∼20-50 RNAPs. Apart from their importance for bacterial transcription, our studies pave the way for molecular-level analysis of several cellular processes at the nanometer scale.

Superresolution microscopy reveals spatial separation of UCP4 and F 0 F 1 -ATP synthase in neuronal mitochondria

“…The localization data were rendered using the Thomson blurring (46). Briefly, each localization is represented as a 2D Gaussian function with a width according to the precision of the respective localization determined from the fitted number of photons and background (45).…”

Investigating Focal Adhesion Substructures by Localization Microscopy

“…However, it is difficult to interpret this result in terms of FA substructure properties, especially considering the heterogeneity in size and shape of the FAs themselves. Such difficulties can be avoided by clustering methods that identify individual clusters based on criteria related to the local density of localizations, such as the nearest neighbor method (30) or density-based spatial clustering of applications with noise (DBSCAN) (31). We applied DBSCAN (32) to the same subset of the PALM data mentioned above (Fig.…”

Colocalization of distant chromosomal loci in space in E. coli: a bacterial nucleolus

“…In previous wide-field studies using RNAP-GFP fusions, it was observed that the fluorescent RNAP foci disappeared, and RNAPs became evenly distributed when rRNA transcription was reduced or inhibited (Cabrera and Jin 2003). Similarly, inhibition of rRNA transcription in single-molecule studies resulted in more evenly distributed RNAPs (Endesfelder et al 2013). Therefore, it was proposed that a nucleolus-like structure might be created by high numbers of RNAPs engaged in rRNA synthesis.…”

“…Several reports in recent years have speculated that there might be nucleolus-like structures in bacteria (e.g., Lewis et al 2000;Cabrera and Jin 2003;Endesfelder et al 2013;Jin et al 2013). Fluorescence-labeled "transcription foci" were observed in fast-growing strains encoding fusions of GFP to RNAP, correlating with the high numbers of RNAP molecules expected to be engaged in transcribing rRNA under these conditions.…”

“…Consistent with the idea that at least some of these fluorescent foci represented rRNA operons, their intensities declined in starved or slowly growing cells. Superresolution imaging showed that RNAPs formed clusters that occupied specific regions of the nucleoid under the conditions examined; i.e., at fast growth rates when there are large numbers of RNAPs actively transcribing rRNA operons (Bratton et al 2011;Bakshi et al 2013;Endesfelder et al 2013;Stracy et al 2015). However, such studies directly addressed only the locations of RNAP and not the positions of the different rRNA operons.…”