Tracking single molecules in uncharted territory : A single-molecule method to study kinetics in live bacteria

Abstract: The synthesis of proteins, also known as translation, is a fundamental process in every living organism. The steps in the translation of genetic information to functional proteins have been meticulously studied, mostly using in vitro techniques, yielding a detailed model of their mechanisms. However, the use of minimal cell-free systems allows for the possibility to miss interactions from absent components or that reactions are affected by the buffer composition. The work presented in this thesis opens a way to study the kinetics of complex molecular processes, like protein synthesis, directly inside live bacterial cells in real time. We developed and optimized a method to deliver dye-labeled macromolecules inside live cells and generate a kinetic model of the particle’s interactions based on its diffusion inside the cell.This method facilitated the study of translation elongation and initiation directly in live cells. Our measurements of reaction times of tRNA in the ribosome, agree with previous reports from in vitro techniques. We further applied the method to examine the effects of three aminoglycoside antibiotics and erythromycin directly in live cells. The aminoglycoside antibiotics slowed-down protein synthesis 2- to 4-fold, while the number of elongation cycles per initiation event decreased significantly. In the case of erythromycin, cells showed a 4-fold slower protein synthesis. Additionally, we measured the kinetics of sequence-specific effects of erythromycin: translational arrest, and peptidyl-tRNA drop-off; these in vivo measurements revealed a complex mechanism of action of the drug, in agreement with models suggested by previous experiments. Additionally, we applied the method to measure the effects, on the kinetics of protein synthesis, caused by modifications in the C-terminal tail of the S13 ribosomal protein. Our measurements showed that specific mutations led to different changes in the occupancy and dwell-time of labeled-tRNA in the ribosome.To summarize, the present work will guide the reader through the development of a method to study the kinetics of protein synthesis directly in live bacterial cells, as well as its application to characterize the effects of different antibiotics within the complex environment of a living organism.