Following Translation by Single Ribosomes One Codon at a Time

摘要

The synthesis of proteins, translation, is carried out by ribosomes. Transla-tion is a dynamic process whereby codons in the messenger RNA (mRNA)are "read" one at a time by base pairing with transfer RNA (tRNA) mole-cules and the corresponding amino acids are polymerized sequentially form-ing polypeptides. To date, the dynamics of translation, in particular themovement of ribosomes along the message as codons are read, and the con-formational changes that ribosomes undergo during the process are not wellunderstood. To quantify the dynamics of translation, the authors tethered anmRNA containing a single hairpin between a micropipette at one end and anoptical trap at the other end; a single ribosome was loaded on the mRNA butheld in place by omitting a tRNA (Fig. 1). Upon resumption of translation byresupplying the tRNA, the investigators could measure the rate of ribosomemovement by the lengthening of the mRNA due to unwinding of the hairpin.The investigators determined that translation occurred in step–pause–steprepeats (Fig. 2), where a pause corresponded to the breaking of three hairpinbase pairs and the translocation of a codon. The coupled unwinding andtranslocation occurred within 0.78 s. In addition, a second longer pause last-ing 1-2 min was also observed near internal Shine Dalgarno sequences, sug-gesting that these "translation arrests" were due to the base pairing betweenthese sequences and the 16S ribosomal RNA. The longer pause appears toplay a regulatory function in translation but might lead to mutational defectsdue to frame shifting as well as protein misfolding. Translation dynamicsalso showed that the translocation time is independent of unwinding, whichrequires force to effect strand separation. While mRNAs are translated com-pletely, translation time by ribosomes depends on the secondary structure ofthe mRNA with hairpin regions translated more slowly than nativelyunfolded regions.