The Dynamics of Eukaryotic Ribosomal Translocation

Abstract

Protein synthesis is an important but complex process that supports life in all domains and takes place in a molecular machine called the ribosome. Numerous atomic structures of ribosome complexes have been resolved and provide snapshots of protein synthesis. Although structural studies describe the global information of translation at the atomic level, they do not report on the dynamic processes of initiation and elongation. The traditional kinetics method provides overall translation dynamics, but it is challenging to study multistep, heterogeneous translation systems. Consequently, the mechanisms and dynamics of translation remain poorly understood. To study real-time protein synthesis, we focus on applying the single-molecule method. Single-molecule Förster resonance energy transfer (smFRET) can track the movement of the ribosome in real-time. We developed a eukaryotic smFRET system that reports on the intersubunit conformation of the ribosome. We employed it to reveal the mechanism of translocation of both internal ribosome entry site (IRES) and tRNA-mRNA translation. Our results demonstrated that the mid and late stages of ribosomal translocation are thermally driven, providing direct evidence that the ribosome is a Brownian molecular motor. Finally, we discuss the potential other applications of the developed system and explore possible directions for expanding current single-molecule toolboxes.