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-...
The ribosome translates the genetic information encoded in messenger RNA into protein. Folded structures in the coding region of an mRNA represent a kinetic barrier that slows the peptide elongation rate, as the ribosome must disrupt structures it encounters in the mRNA at its entry site to enable translocation to the next codon. Such structures are exploited by the cell to create diverse strategies for translation regulation, such as programmed frameshifting1,2, protein expression levels3,4, ribosome localization5, and cotranslational protein folding6. Although strand separation activity is inherent to the ribosome, requiring no exogenous helicases7, its mechanism is still unknown. Here, using a single-molecule optical tweezers assay on mRNA hairpins, we find that the translation rate of identical codons at the decoding center is greatly influenced by the G•C content of folded structures at the mRNA entry site. Furthermore, force applied to the ends of the hairpin to favor its unfolding significantly speeds translation. Quantitative analysis of the force dependence of its helicase activity reveals that the ribosome, unlike previously studied helicases, uses two distinct active mechanisms to unwind mRNA structure: (i) it destabilizes the helical junction at the mRNA entry site by biasing its thermal fluctuations toward the open state, increasing the probability for the ribosome to translocate unhindered; and (ii) it also mechanically pulls apart the mRNA single-strands of the closed junction during the conformational changes that accompany ribosome translocation. Our results establish a quantitative mechanical basis for understanding the mechanism of regulation of the elongation rate of translation by structured mRNAs.
Circular dichroism (CD) spectroscopy is widely used to characterize the structures of DNA G-quadruplexes. CD bands at 200-300 nm have been empirically related to G-quadruplexes having parallel or antiparallel sugar-phosphate backbones. We propose that a more fundamental interpretation of the origin of the CD bands is in the stacking interactions of neighboring G-quartets, which can have the same or opposing polarities of hydrogen bond acceptors and donors. From an empirical summation of CD spectra of the d(G)5 G-quadruplex and of the thrombin binding aptamer that have neighboring G-quartets with the same and opposite polarities, respectively, the spectra of aptamers selected by the Ff gene 5 protein (g5p) appear to arise from a combination of the two types of polarities of neighboring G-quartets. The aptamer CD spectra resemble the spectrum of d(G3T4G3), in which two adjacent quartets have the same and two have opposite polarities. Quantum-chemical spectral calculations were performed using a matrix method, based on guanine chromophores oriented as in d(G3T4G3). The calculations show that the two types of G-quartet stacks have CD spectra with features resembling experimental spectra of the corresponding types of G-quadruplexes.
Experimental variables of optical tweezers instrumentation that affect RNA folding/unfolding kinetics were investigated. A model RNA hairpin, P5ab, was attached to two micron-sized beads through hybrid RNA/DNA handles; one bead was trapped by dual-beam lasers and the other was held by a micropipette. Several experimental variables were changed while measuring the unfolding/refolding kinetics, including handle lengths, trap stiffness, and modes of force applied to the molecule. In constant-force mode where the tension applied to the RNA was maintained through feedback control, the measured rate coefficients varied within 40% when the handle lengths were changed by 10-fold (1.1-10.2 Kbp); they increased by two- to threefold when the trap stiffness was lowered to one-third (from 0.1 to 0.035 pN/nm). In the passive mode, without feedback control and where the force applied to the RNA varied in response to the end-to-end distance change of the tether, the RNA hopped between a high-force folded-state and a low-force unfolded-state. In this mode, the rates increased up to twofold with longer handles or softer traps. Overall, the measured rates remained with the same order-of-magnitude over the wide range of conditions studied. In the companion article on pages 3010-3021, we analyze how the measured kinetics parameters differ from the intrinsic molecular rates of the RNA, and thus how to obtain the molecular rates.
Summary Programmed ribosomal frameshifting produces alternative proteins from a single transcript. -1-frameshifting occurs on Escherichia coli’s dnaX mRNA containing a slippery sequence AAAAAAG and peripheral mRNA structural barriers. Here we reveal hidden aspects of the frameshifting process, including its exact location on the mRNA and its timing within the translation cycle. Mass spectrometry of translated products shows that ribosomes enter the -1-frame from not one specific codon but various codons along the slippery sequence and slip by not just -1 but also -4, or +2-nucleotides. Single-ribosome translation trajectories detect distinctive codon-scale fluctuations in ribosome-mRNA displacement across the slippery sequence, representing multiple ribosomal translocation attempts during frameshifting. Flanking mRNA structural barriers mechanically stimulate the ribosome to undergo back-and-forth translocation excursions, broadly exploring reading frames. Both experiments reveal aborted translation around mutant slippery sequences, indicating that subsequent fidelity checks on newly adopted codon position base-pairings lead to either resumed translation or early termination.
RNA unfolding and folding reactions in physiological conditions can be facilitated by mechanical force one molecule at a time. By using force-measuring optical tweezers, we studied the mechanical unfolding and folding of a hairpin-type pseudoknot in human telomerase RNA in a near-physiological solution, and at room temperature. Discrete two-state folding transitions of the pseudoknot are seen at ;10 and ;5 piconewtons (pN), with ensemble rate constants of ;0.1 sec À1 , by stepwise force-drop experiments. Folding studies of the isolated 59-hairpin construct suggested that the 59-hairpin within the pseudoknot forms first, followed by formation of the 39-stem. Stepwise formation of the pseudoknot structure at low forces are in contrast with the one-step unfolding at high forces of ;46 pN, at an average rate of ;0.05 sec À1 . In the constant-force folding trajectories at ;10 pN and ;5 pN, transient formation of nonnative structures were observed, which is direct experimental evidence that folding of both the hairpin and pseudoknot takes complex pathways. Possible nonnative structures and folding pathways are discussed.
A detailed understanding of tRNA/mRNA translocation requires measurement of the forces generated by the ribosome during this movement. Such measurements have so far remained elusive and, thus, little is known about the relation between force and translocation and how this reflects on its mechanism and regulation. Here, we address these questions using optical tweezers to follow translation by individual ribosomes along single mRNA molecules, against an applied force. We find that translocation rates depend exponentially on the force, with a characteristic distance close to the one-codon step, ruling out the existence of sub-steps and showing that the ribosome likely functions as a Brownian ratchet. We show that the ribosome generates ∼13 pN of force, barely sufficient to unwind the most stable structures in mRNAs, thus providing a basis for their regulatory role. Our assay opens the way to characterizing the ribosome's full mechano–chemical cycle.DOI: http://dx.doi.org/10.7554/eLife.03406.001
By exerting mechanical force, it is possible to unfold/refold RNA molecules one at a time. In a small range of forces, an RNA molecule can hop between the folded and the unfolded state with force-dependent kinetic rates. Here, we introduce a mesoscopic model to analyze the hopping kinetics of RNA hairpins in an optical tweezers setup. The model includes different elements of the experimental setup (beads, handles, and RNA sequence) and limitations of the instrument (time lag of the force-feedback mechanism and finite bandwidth of data acquisition). We investigated the influence of the instrument on the measured hopping rates. Results from the model are in good agreement with the experiments reported in the companion article. The comparison between theory and experiments allowed us to infer the values of the intrinsic molecular rates of the RNA hairpin alone and to search for the optimal experimental conditions to do the measurements. We conclude that the longest handles and softest traps that allow detection of the folding/unfolding signal (handles approximately 5-10 Kbp and traps approximately 0.03 pN/nm) represent the best conditions to obtain the intrinsic molecular rates. The methodology and rationale presented here can be applied to other experimental setups and other molecules.
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