Quantitative Studies of Transcription in E.coli With Subdiffraction Fluorescence Microscopy

Abstract

The organization of biomolecules into macromolecular assemblies is often closely related to biomolecular function. However, such structures often remain unresolved using conventional light microscopy. By applying novel high-resolution single-molecule fluorescence techniques, it becomes possible to study biomolecular structure and interaction below the diffraction limit of light, reaching a lateral resolution of ∼20 nm [1, 2]. We use photoswitchable and photoactivatable fluorescent probes in combination with direct stochastic optical reconstruction microscopy (dSTORM) [2] and photoactivation-localization microscopy (PALM) [3]. Following light-induced activation of a subset of fluorescent probes attached to target proteins, the fluorescent state is read out and single emitters are localized with nanometer precision. This procedure is repeated many times, and the ensemble of coordinates is used to reconstruct an image with superior resolution [2, 3].RNA polymerases have been localized in bacteria using conventional approaches [4]. In our study, we focus on the spatial organization of bacterial transcription sites in E. coli at the molecular scale. To reach that goal, we apply high-resolution fluorescence methods, and we will present a refined understanding of structure and function in the bacterial transcription machinery.