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Light, camera, action: watching single DNA/RNA polymerases at work in living cells

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If you have a question about this talk, please contact Dr Eileen Nugent.

Most single-molecule experiments are performed in vitro, using tightly controlled conditions and well-defined concentrations of a limited number of interacting components. However, to understand biological mechanisms as they occur in vivo while maintaining the extra information provided by single-molecule detection, there is a need for performing such experiments in cellular contexts, and in particular in living cells.

Towards this goal, we have been single-molecule fluorescence to perform localization-based super-resolution imaging (specifically photoactivated localization microscopy, PALM ) and to study the subcellular localization, diffusion properties, abundance and clustering of DNA -binding proteins in bacterial cells. We have also developed physical methods for delivering fluorescent biomolecules in bacteria and observing single-molecule fluorescence and single-molecule FRET in the bacterial cytoplasm.

I will discuss PALM studies of the subcellular localization and copy number of RNA polymerase in bacterial cells grown under different physiological conditions, both in fixed and living bacterial cells; using this method, we have identified the spatial distribution of an immobile fraction that represents mainly RNAP polymerases engaged in promoter and transcribed regions.

I will also how describe how PALM -based single-molecule diffusion tracks of polymerases in living bacteria (middle and bottom panels) allowed us to visualize DNA -damage and DNA -repair processes in real-time and at the single-molecule level for the first time.

Our approaches should be useful for studying many processes in gene replication, expression, and maintenance in various types of cells.

This talk is part of the BSS Formal Seminars series.

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