Single-molecule studies of tRNA dynamics during ongoing translation

The pre-translocation complex of the ribosome can undergo spontaneous fluctuations of mRNA and tRNAs between classical and hybrid states, and occupation of the hybrid tRNA positions has been proposed to precede translocation. The classic and hybrid state tRNA positions been extensively characterized when the ribosome is stalled along the messenger RNA by either the absence or the delayed addition of elongation factor G (EF-G), or by the presence of antibiotics or GTP analogs that block translocation. Surprisingly, during multiple ongoing elongation cycles when both EF-G and ternary complexes are present, we found that tRNA positions in PRE complex ribosome do not fluctuate. Instead, they adopt a stationary intermediate structure between the stalled classical and hybrid tRNA positions, as indicated by single molecule fluorescence resonance energy transfer (FRET) between adjacent tRNAs and between A-site tRNA and ribosomal protein L11. These results indicate that EF-G promotes the formation of an intermediate structure during ongoing translation. smFRET experiments using labeled EF-G at those concentrations requires zero-mode waveguides (ZMWs), arrays of nanoscale holes in a thin metal film that confine the observation volume to reduce prohibitive background fluorescence. However, the need for specialized nanofabrication equipment has precluded their widespread adoption by the biochemical community. In contrast, nanosphere lithography uses the self-assembly of polystyrene beads into a hexagonal array, forming a natural lithographic mask for the deposition of metallic posts in the interstices between beads, followed by a different metal cladding around the posts and dissolution of the posts to leave a well array. The cross-sectional size of those interstices (and thus subsequent posts and wells) can be finely tuned by fusing the beads at the polystyrene glass transition temperature. That bead lattice serves as a template for creating sub-wavelength ZMWs with little specialized equipment and at a low cost, enabling wide-scale adoption