Following translation by single ribosomes one codon at a time

Abstract

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.Current understanding of the ribosome and the mechanism of translation has been significantly strengthened and expanded by recent advances in crystallography 1-6 and cryo-electron microscopy 7-10 . The ribosome undergoes several dynamical structural changes as it moves relative to the mRNA and transfer RNAs during translation 8,11 . Kinetic experiments have given a quantitative description of some of these dynamics during the main steps of the elongation cycle of protein synthesis 12 . During elongation, the secondary structures present in all mRNAs are disrupted to allow movement of the mRNA through the 30S subunit, and the reading of each codon. This task is aided by the mRNA helicase activity of the ribosome that has been localized to the downstream tunnel of the 30S subunit 13 . Moreover, interactions of mRNA pseudoknots or hairpins with the helicase region of the ribosome can shift the reading frame of the mRNA to the -1 frame, and play an important role in regulating gene expression in retroviruses 14-16 . It is extremely difficult to follow the steps of ribosomes during translational elongation using ensemble methods, because the dynamics of individual ribosomes are stochastic 17,18 and it is impossible to synchronize their activity. Here, we have used optical tweezers to follow the step-by-step translation of a single hairpin-forming mRNA molecule by a single ribosome. This approach has allowed us to characterize the dynamics of ribosome translation, measuringCorrespondence and requests for materials should be addressed to I.T. (intinoco@lbl.gov).. the time the ribosome spends at each codon, the number of mRNA nucleotides that move through the ribosome in each translocation step, and the time required per step. We have also determined the effects of mRNA structure on step size and rate, and have studied the effects of internal Shine-Dalgarno sequences 19 on translation arrest. These experiments provide a dynamic picture of the movement of a messenger RNA through a ribosome. NIH Public AccessIn these experiments, we used a single mRNA hairpin with a ribosome stalled at the 5′ end by omission of a required aminoacyl-tRNA; the RNA was attached to two micrometre-...

Stochastic kinetics of ribosomes: Single motor properties and collective behavior

“…On the other hand, it has been claimed [6] that difference of two exponentials fit the experimentally measured f (t) very well. We reconcile these two observations by identifying the parameter regime where our theoretically derived f (t) is, indeed, well approximated by difference of two exponentials [7,8,9,10,11].…”

“…The sum of the durations of a pause and the following translocation defines the time of a dwell at the corresponding codon. Recently, using an ingenious method, the distribution f (t) of the dwell times of a ribosome has been measured [6]. We present a systematic derivation of this distribution from a detailed kinetic theory of translation which incorporates the mechanochemical cycle of individual ribosomes.…”

Accurate Prediction of Cellular Co-Translational Folding Indicates Proteins can Switch from Post- to Co-Translational Folding

“…The covalent attachment of amino acids into polypeptides is a many-step process 55 . However, a two-exponential fit of the experimentally-measured ribosome dwell-time distribution indicates only two rate limiting steps 13,56 . Therefore, to numerically test if the predicted co-translational folding curve would change significantly when a dwell-time distribution of the form PðtÞ ¼ k1k2 k2…”

Nanomanipulation of Single RNA Molecules by Optical Tweezers

“…With many improvements, the Minitweezers display extraordinary stability as compared to the first generation optical tweezers. A number of research groups in several countries use the Minitweezers in their single-molecule research 14,15,[34][35][36][37] . The details of construction, calibration, and operation of the device, including instruction videos, are available at the "Tweezers Lab" website (http://tweezerslab.unipr.it).…”

“…The technique has been employed to study folding and mechanics of DNA [4][5][6] , RNA [7][8][9] , and proteins 10,11 . Optical tweezers have also been used to observe DNA replication 12 , RNA transcription 13 , and protein synthesis 14,15 , as well as many other biomolecular events [16][17][18] .…”