Following translation by single ribosomes one codon at a time

We have followed individual ribosomes as they translate single messenger RNA hairpins tethered by the ends to optical tweezers. Here we reveal that translation occurs through successive translocation-and-pause cycles. The distribution of pause lengths, with a median of 2.8 s, indicates that at least two rate-determining processes control each pause. Each translocation step measures three bases—one codon—and occurs in less than 0.1 s. Analysis of the times required for translocation reveals, surprisingly, that there are three substeps in each step. Pause lengths, and thus the overall rate of translation, depend on the secondary structure of the mRNA; the applied force destabilizes secondary structure and decreases pause durations, but does not affect translocation times. Translocation and RNA unwinding are strictly coupled ribosomal functions. Found in translation Recent advances in crystallography and cryo-electron microscopy have increased our current understanding of the ribosome's role in protein synthesis, the translation of the information encoded by messenger RNA code into a polypeptide. Now in a technical tour de force , Jin-Der Wen et al . have used optical tweezers to follow the translation of a single mRNA by an E. coli ribosome one codon at a time. An mRNA (yellow on the cover) is tracked as it is unwound and translated. Translation, it turns out, is not a continuous process but occurs through successive translocation-and-pause cycles. Each cycle consists of a translocation step of three nucleotides in less than 0.1 s, then a pause of a few seconds. These are the first direct real-time observations of the physical steps of the ribosome machine along the mRNA during translation. The resulting single-molecule assay can address problems in translation not accessible using traditional bulk methods, including translational gene regulation and the fidelity of translation. Graphic by Laura Lancaster and Courtney Hodges, using RIBBONS (M Carson, 1997). Use of a single-molecule approach finds that translation by the ribosome occurs through a reiterative process of translocation and pausing. A single translocation event covers three nucleotides, in which three substeps are detected, and is rapid. The pauses, which are longer and whose length depends on the mRNA's secondary structure, are rate-determining and are composed of at least two processes.