Single-Molecule Fluorescence Studies of Nucleic-Acid Transactions in Living Bacteria

Abstract

Most single-molecule fluorescence experiments are performed in vitro, using tightly controlled conditions and well-defined concentrations of few 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 single-molecule experiments in cellular contexts, and in particular in living cells.Towards this goal, we have developed general localization-based super-resolution imaging methods (specifically, based on photo-activated localization microscopy, PALM) to study the subcellular localization, diffusion properties, abundance and clustering of DNA-binding proteins in bacterial cells. We have also developed general physical methods for delivering fluorescent biomolecules (such as DNA, RNA, and proteins) in living bacteria and performing single-molecule tracking and single-molecule FRET studies in the bacterial cytoplasm.I will discuss examples of applications of single-molecule tracking towards understanding the subcellular localization and intracellular mobility of many proteins interacting with DNA and RNA substrates; I will also present examples of using single-molecule FRET to study the conformations of such nucleic-acid substrates as they are being processed in vivo. The examples will include studies of gene transcription, RNA translation and DNA repair in living Escherichia coli cells.