Single-Molecule Profiling of Ribosome Translational Phenomena

Abstract

Translation elongation is a heterogeneous process, involving multiple compositional factors stochastically binding to the ribosome to direct protein synthesis, which in turn regulates the conformation of the ribosome. The rate of translation is often regulated by the underlying messenger RNA (mRNA) sequence and structure. Here, we use single-molecule fluorescence resonant energy transfer (FRET) and colocalization with zero-mode waveguides (ZMWs) to correlate directly ribosome conformations and compositions of thousands of ribosomes simultaneously during multiple rounds of elongation. This allows us to profile global translational rates while delineating mechanistic details of the dynamics with codon resolution. We first studied translation of a canonical mRNA with uniform translation rates to establish the tight interplay between compositional factors and conformational dynamics of the ribosome during elongation. We then determined how mRNA sequences and structures, such as hairpins and possible ribosome-mRNA base pairing, as expressed in the the dnaX −1 frameshifting sequence, perturb the basal elongation process. Our results show how mRNAs can modulate and uncouple ribosomal conformational and compositional dynamics.