Super-Resolution Imaging of Transcription in Live Bacterial Cells

Abstract

While we can measure average gene expression levels with bulk biochemistry assays, much less is known about where transcription occurs within individual cells and how this varies in response to changes in the cells' environment. Here we use photoactivated localization microscopy (PALM) combined with single particle tracking to directly visualise individual fluorescently labelled RNA polymerases in live Escherichia coli. Following on from previous studies in fixed cells1, this work in live cells allows us to track the motion of single molecules and analyse their diffusive motion. After accounting for fluorophore blinking, we find that between 45 - 50% of RNA polymerases have an apparent diffusion coefficient of less than 0.1 μm2s−1. These molecules are likely to be bound to DNA on promoters or transcribing genes, and hence tell us where gene expression is taking place inside living cells.Under different growth conditions the ratio of bound to diffusing molecules remains similar; however, there are clear changes in the spatial distribution of bound molecules. In rich media, immobile RNA polymerases show large clusters of between 40 and 80 molecules, likely due to heavy transcription on the seven E. coli ribosomal operons. This is the first time such clustering has been seen in live cells. In nutrient poor media extensive clustering is not seen, consistent with a wider distribution of transcription over many genes.Comparing the spatial distributions of bound and diffusing RNA polymerases also tells where transcription happens relative to the nucleoid. Averaging over many cells show that bound RNA polymerases are more likely to be found at the nucleoid periphery, indicating that the bacterial chromosome may be spatially organised with transcribed regions close to the cytoplasm.1. Endesfelder, U. et al. Biophysical Journal105, 172-181 (2013).