Spatial organization of RNA polymerase and its relationship with transcription in Escherichia coli

Abstract

Recent studies have shown that RNA polymerase (RNAP) is organized into distinct clusters in Escherichia coli and Bacillus subtilis cells. Spatially organized molecular components in prokaryotic systems imply compartmentalization without the use of membranes, which may offer insights into unique functions and regulations. It has been proposed that the formation of RNAP clusters is driven by active ribosomal RNA (rRNA) transcription and that RNAP clusters function as factories for highly efficient transcription. In this work, we examined these hypotheses by investigating the spatial organization and transcription activity of RNAP in E. coli cells using quantitative superresolution imaging coupled with genetic and biochemical assays. We observed that RNAP formed distinct clusters that were engaged in active rRNA synthesis under a rich medium growth condition. Surprisingly, a large fraction of RNAP clusters persisted in the absence of high rRNA transcription activities or when the housekeeping σ70 was sequestered, and was only significantly diminished when all RNA transcription was inhibited globally. In contrast, the cellular distribution of RNAP closely followed the morphology of the underlying nucleoid under all conditions tested irrespective of the corresponding transcription activity, and RNAP redistributed into dispersed, smaller clusters when the supercoiling state of the nucleoid was perturbed. These results suggest that RNAP was organized into active transcription centers under the rich medium growth condition; its spatial arrangement at the cellular level, however, was not dependent on rRNA synthesis activity and was likely organized by the underlying nucleoid.

Clusters of bacterial RNA polymerase are biomolecular condensates that assemble through liquid-liquid phase separation

“…RNAP is distributed throughout the nucleoid in cells grown in minimal media, but concentrates into distinct clusters in cells grown in rich media (31,32). This nutrient-dependent localization has been well-characterized (33,34), but the mechanism(s) that govern RNAP clustering remains unclear (35,36). Here, we show that clusters of bacterial RNAP are biomolecular condensates.…”

“…These results contradict expectations for the DNA-binding model, but are fully consistent with an LLPS model in which phase separation is mediated primarily by protein-protein interactions, rather than RNA-protein interactions. Furthermore, the size of RNAP clusters is smaller after disruption of rRNA transcription (36). This behavior is similar to the nucleolus, which is stabilized by rRNA, but whose protein components nevertheless condense into small droplets in the absence of rRNA transcription in early C. elegans embryos (17).…”

“…Under fast growth conditions, RNAP is densely packed on rrn operons (46) and the vast majority are engaged in synthesis of ribosomal RNA (rRNA) (47), prompting comparisons to the eukaryotic nucleolus (37). However, recent evidence indicates that nucleoid structure may play a more dominant role in RNAP clustering than transcriptional activity itself (36). Alternatively, we hypothesize that RNAP clusters may instead assemble primarily by LLPS such that RNAP is maintained at a high local concentration through weak multivalent protein-protein interactions, independent of its transcriptional activity ( Fig.…”

“…Furthermore, these estimates are consistent with Klumpp's numerical model (97), as well as previous singlemolecule studies. PALM analyses conducted at slower growth rates (31 -73 min doubling times) counted between 70 and 500 molecules per cluster and 2 to 4 clusters per cell (33,34,36), out of a total of ~5000 RNAPs per cell. Therefore, at both fast and intermediate growth conditions, less than half of all RNAPs are engaged in rRNA transcription and we propose that some of the nontranscribing pool is partitioned into condensates.…”

Vibrionaceae core and shell genes are non-randomly distributed into spatially distinct intracellular domains

“…The non-random structural organization of the genes suggests to us that there is a strong link between gene placement and their function, and that the underlying reasons for the strong distribution pattern could be very complex. The full complexity of factors that affects gene expression can be illustrated by e.g., chromatin packing [37][38][39][40][41], nucleoid-associated proteins (NAPs) [42][43][44], Structural Maintenance of Chromosome complex (SMC) [45], RNA polymerase (RNAP) [46][47][48][49][50], transcription factors and promoter strength/chromosomal position [43,51] and macromolecular crowding [20]. Perhaps the most fundamental factor is chromatin packing and organization.…”