Single-molecule observations of ribosome function

Abstract

Single-molecule investigations promise to greatly advance our understanding of basic and regulated ribosome functions during the process of translation. Here, recent progress towards directly imaging the elemental translation elongation steps using fluorescence resonance energy transfer (FRET)-based imaging methods is discussed, which provide striking evidence of the highly dynamic nature of the ribosome. In this view, global rates and fidelities of protein synthesis reactions may be regulated by interactions of the ribosome with mRNA, tRNA, translation factors and potentially many other cellular ligands that modify intrinsic conformational equilibria in the translating particle. Future investigations probing this model must aim to visualize translation processes from multiple structural and kinetic perspectives simultaneously, to provide direct correlations between factor binding and conformational events.

Figure 1. When placed in informative positions, FRET Pairs on the ribosome provide a “molecular EKG” reporting on transitions between discrete conformational states

(A) Top-down view of an atomic representation of the two-subunit e.coli ribosome (derived from PDBIDs:1nkw;1ibm; and 486d), rendered in Pymol. The mRNA track (grey), A-, P- and E-site tRNA binding sites (red) as well as sites fluorescently labeled in the FRET studies discussed are highlighted. As diagrammed, aminoacyl-tRNA (aa-tRNA) enter the leading edge of the ribosome at the A site and transit the P and E sites before dissociating from the particle. Oligonucleotide tagging sites utilized by Marshall et al., cannot be seen here as both sites lie at the base of the ribosomal subunits opposite the illustrated vantage point. (B) A single-molecule fluorescence trajectory (green=donor; red=acceptor) and FRET trajectory (blue) obtained using a wide-field TIR imaging system at 40ms time resolution under equilibrium conditions from a surface-immobilized ribosome bearing site-specifically labeled A- and P-site tRNA molecules in the absence of energy input or translation factors[35]. The dynamic FRET data, reporting directly on thermally-accessible, nanometer-scale changes in tRNA position within the ribosome, persist until donor and/or acceptor fluorophore photobleaching. The recording shown is exemplary in that the duration of fluorescence extends for more than one minute.