Spatial Organization of Transcription in E. Coli using Super-Resolution Fluorescence Microscopy

Abstract

Transcription, the process of converting genetic information stored in DNA to RNA, lies at the heart of gene expression. Transcription has been studied extensively in-vitro to probe its mechanistic detail, however, these conditions differ from the complex environment inside a living cell. Spatial distributions of molecular components have recently been shown to be an important facet of gene regulation in prokaryotic systems. We investigated the spatial distributions of various molecular components of transcription in E. coli (genes, RNA Polymerase (RNAP), elongation factors, nascent transcripts), and their physical correlation with each other, to gain insight into the regulation of gene expression at the global, cellular level. Previously, it has been shown that E. coli RNAP form a few clusters per cell under fast growth. We used super-resolution fluorescence microscopy to investigate RNAP distribution under various growth conditions to better characterize their spatial patterns. We found that RNAP forms distinct clusters under fast growth that are largely retained in cells put under different global transcription regimes (i.e. stringent response, inhibition of gyrase). RNAP clusters are the most homogeneously distributed in cells without active transcription via rifampicin treatment. Additionally, we used multi-color super-resolution imaging to correlate the spatial localization of RNAP clusters with DNA sites, nascent rRNA and elongation factor NusA to further elucidate the underlying make-up of RNAP clusters. Our results show that RNAP clusters are highly co-localized with NusA, thus are likely composed of elongation complexes. Interestingly, while RNAP clusters have a relatively high level of colocalization with nascent rRNA, and rrn DNA sites in fast growing cells, RNAP clusters are retained under conditions where the level of rRNA synthesis is reduced (SHX, deletion of rrn), this indicates a connection between rRNA synthesis and RNAP clusters, but also points to the independence of formation of RNAP clusters from active rRNA synthesis.