Live-cell Single-molecule Tracking for Protein Synthesis Kinetics Measurements

Abstract

Our ability to relate results from biochemical experiments in the test tube, to the dynamic processes occurring in living cells is very limited. We have recently developed new experimental and analytical tools to directly study the kinetics of fast biochemical reactions in live cells. In a pioneering study, dye-labeled elongator tRNAPhe or initiator tRNAfMet were electroporated into E. coli cells, and tracked using super-resolved single-molecule microscopy. Using Hidden Markov Model based analysis of the diffusion trajectories, we estimated the dwell time of the tRNAs bound to ribosomes. Hence, with this approach we were able to directly measure kinetics of translation initiation and elongation at codon resolution inside living cells. In a follow-up study, the system was used to investigate the direct effect of the antibiotic chloramphenicol on ongoing protein synthesis. In this presentation, I will talk about our method, and in particular how we are now applying the method to study related biological phenomena. For example, by tracking key components of the SRP (Signal Recognition Particle) pathway, we measure the kinetics of co-translational translocon targeting of inner-membrane proteins.