Single-Cell Biophysics: Measurement, Modulation, and Modeling

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Single-Cell Biophysics: Measurement, Modulation, and Modeling Poster Abstracts
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95-POS Board 48
Spatial Organization of Transcription in E. coli via Superresolution Fluorescence
Microscopy
Xiaoli Weng1, Christopher H. Bohrer1, Arvin C. Lagda2, Jie Xiao1.
1Johns Hopkins School of Medicine, Baltimore, MD, USA, 2 Icahn School of Medicine at Mount
Sinai, New York, NY, USA.
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 and their physical
correlation with each other, to gain insight into the regulation of gene expression at the global,
cellular level. Using superresolution fluorescence microscopy, we found that RNA Polymerase
(RNAP) forms distinct clusters under fast growth that are largely retained in cells under different
global transcription perturbations. RNAP clusters are the most homogenously distributed in cells
without active transcription via rifampicin treatment. Additionally, we used multi-color
superresolution 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 certain level of
colocalization with nascent rRNA, and rrn DNA sites in fast growing cells, RNAP clusters are
retained under conditions where rRNA synthesis is reduced. This points to the independence of
formation of RNAP clusters from active rRNA synthesis. These results suggest a high level of
heterogeneity both in the spatial organization and functional role of RNAP clusters in E. coli.