Investigation of Ribosomal Dynamics in E.coli Using Single-molecule FRET

Abstract

Translation, the last step of gene expression, highly determines an organism’s normal function. Ribosomes are molecular motors that synthesize proteins from mRNA using amino acids carried by tRNAs. A ribosome’s mobility on mRNA is provided by conformational changes such as 30S subunit head movements, L1 stalk movements, and intersubunit rotations. Intersubunit rotations are the counterclockwise and clockwise rotations that happen between the large and small ribosomal subunits. Major steps of translation such as ribosome assembly, peptidyl transfer, and translocation are promoted by intersubunit rotations. However, ribosome rotations are transient, making these movements difficult to capture. Here, high-speed single-molecule microscopy was used to follow the translational intersubunit rotations in real time. The 30S subunit was labeled with a fluorescent donor dye (Cy3B) on helix 44 and the 50S subunit was labeled with an acceptor dye (Cy5) on helix 101. The FRET changes between two labeled subunits were used to study the translation mechanism by monitoring the ribosome’s rotations. First, mRNA initiation was studied using fluorescently labeled leaderless mRNA which demonstrated that leaderless mRNA could initiate through two pathways. Next, the antibiotics viomycin, neomycin, and spectinomycin were added to perturb translation by disrupting ribosome movements. This further revealed the mechanism of initiation. Finally, ribosomes were found to spontaneously exchange between three different rotational conformations in the pre-translocation state. These results suggest that ribosome rotations are highly involved with translation. Understanding translational mechanisms will contribute to understanding bacterial gene expression, stress adaptation, and the evolution of protein synthesis.