Author response: Direct measurement of the mechanical work during translocation by the ribosome (2014)

Article Figures and data Abstract eLife digest Introduction Results and discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract 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. https://doi.org/10.7554/eLife.03406.001 eLife digest Producing a protein first requires its gene to be transcribed into a long molecule called a messenger RNA (mRNA). A complex molecular machine called the ribosome then translates the mRNA code by reading it three letters at a time. Each triplet of letters—known as a codon—tells the ribosome which amino acid to add next into the protein. After adding an amino acid, the ribosome moves along the mRNA molecule to read the next codon and add another amino acid into the protein chain. While researchers understand how protein chains are formed, how the ribosome shifts along the mRNA strand—a process called translocation—is still unclear. It is known that this process involves many force-generating movements and changes to the shape of the ribosome. However, it is only recently that researchers have been able to measure these forces. Using optical tweezers—an instrument that uses a highly focused laser beam to hold and manipulate microscopic objects—Liu, Kaplan et al. followed individual ribosomes as they translated an mRNA and measured the effect that applying an opposing force has on the rate of translation. The results shed new light on the mechanism of translocation. First, Liu, Kaplan et al. found that ribosomes jump directly from one triplet to the next in the mRNA sequence, rather than moving there in a series of smaller steps. Next, the results indicate that translocation occurs spontaneously, driven by thermal energy, while chemical reactions prevent the reverse movement, in a mechanism known as a 'Brownian Ratchet'. Measurements of the maximum force generated by the ribosome also give insights into how translation is regulated. Strands of mRNA can fold into certain structures that slow down translation, because the mRNA must first be unfolded before the ribosome can translate it. Liu, Kaplan et al. found that the maximum force generated by a ribosome is only just enough to unwind these mRNA structures, making the translation rate highly sensitive to the existence of such structures, and the structures themselves of high importance for regulating transcription. Given its importance as the ultimate decoder of the genetic information, understanding the ribosome's function and regulation has broad implications. The work of Liu, Kaplan et al. opens the way for a full characterization of the role of mechanical forces in the translation process. https://doi.org/10.7554/eLife.03406.002 Introduction Ribosomes possess three binding sites for tRNA: the aminoacyl (A), peptidyl (P), and exit (E) sites, each of which is shared between the 30S and 50S ribosomal subunits. Following codon recognition and peptide bond formation, the ribosome has a deacylated tRNA in the P site and a peptidyl-tRNA in the A site. In order to start a new elongation cycle, the A site must be emptied to allow binding of the next aminoacyl-tRNA. To this end, the tRNAs and mRNA must move relative to the ribosome. This movement occurs in two steps (Moazed and Noller, 1989b): first, the 3′ ends of the tRNAs in the A and P sites move, with respect to the 50S subunit, into hybrid A/P and P/E states, respectively. In vitro, formation of these states can occur spontaneously, reversibly, and independently of elongation factor G (EF-G) (Moazed and Noller, 1989b; Sharma et al., 2004; Cornish et al., 2008; Munro et al., 2010) and is coupled to rotation of the 30S body (Moazed and Noller, 1989b; Frank and Agrawal, 2000; Agirrezabala et al., 2008; Julian et al., 2008; Dunkle et al., 2011). In the second step, which is irreversible and EF-G-dependent (Moazed and Noller, 1989b; Savelsbergh et al., 2003), the mRNA is translocated by one codon, along with movement of the associated anticodon ends of the tRNAs to the classical P and E sites, coupled to an orthogonal rotation of the 30S subunit head domain (Ratje et al., 2010; Dunkle et al., 2011; Ermolenko and Noller, 2011; Guo and Noller, 2012; Zhou et al., 2013). The translocation process also involves other large-scale conformational changes in the ribosome, including reverse rotational movements of the 30S subunit body and head (Ermolenko and Noller, 2011; Guo and Noller, 2012), and movement of the large subunit L1 stalk into the intersubunit space (Fei et al., 2008; Cornish et al., 2009). Translocation is therefore a highly coordinated and complex process composed of inter- and intra-molecular, force-generating mechanical movements. During translation, the ribosome must also overcome significant mechanical barriers posed by structured portions of the mRNA. These structures are exploited by the cell to create diverse strategies for translation regulation; for example, pseudoknots and hairpins are used to induce programmed frameshifting (Tsuchihashi, 1991; Namy et al., 2006), whereas synonymous mutations that can alter the local structure of RNA and codon usage are employed to control protein expression levels (Duan et al., 2003; Nackley et al., 2006). Hence, force is not only a product of the chemical reactions during translation, but also an important player in the regulation of this process. Results and discussion We have designed an experiment to monitor the movement of individual ribosomes against an opposing force during translation (Figure 1A). A gene fusion encoding ribosomal protein S16 linked via its C-terminus to the biotinylation domain of the biotin carboxyl carrier protein (BCCP) was introduced (Link et al., 1997) into the chromosome of Escherichia coli, and biotinylated ribosomes were then purified. In the presence of initiation factors, initiator tRNA and GTP, a biotinylated ribosome is assembled at the AUG start site of an mRNA, whose 3′ end had been previously annealed to a complementary DNA handle harboring a 5′ digoxigenin. The complex is then tethered between a pair of 2.1 µm diameter polystyrene beads: a streptavidin-coated bead, which binds to the ribosome and is held by suction on the end of a micropipette, and an anti-digoxigenin antibody-coated bead, which binds to the DNA handle and is held in an optical trap. Next, a mixture containing elongation factors, aminoacyl-tRNAs and GTP is introduced into the experimental chamber. The tension in the tether, stabilized by an automated feedback routine, produces a constant opposing force as translocation proceeds. Translation is followed in real time as a decrease in the tether length between the beads (Figure 1B–C, Figure 1—figure supplement 1). No translation signals were detected in the absence of GTP and EF-G. (Figure 1E,F). Figure 1 with 1 supplement see all Download asset Open asset Following translation by a single ribosome on a single mRNA. (A) Geometry for single-molecule translation experiments. A biotinylated ribosome is loaded onto a single-stranded mRNA and attached to a streptavidin-coated polystyrene bead fixed to a micropipette. The 3′ of the message is anchored to a second bead through a 1460 bp DNA/RNA hybrid handle. Calibrated forces can be applied to the ribosome by manipulating the second bead with an optical trap, while the translation progress of the ribosome is determined by the change in extension of the tether. (B–D) Typical translation events recorded under 4, 6 and 8 pN of constant tension. The upper panels show the codons translated as a function of time, and indicate that translation proceeds not in a continuous manner, but in a series of translational bursts separated by long pauses. The gray line shows the raw (1 kHz) data, while green (translocation) and red (pause) are filtered down to 1 Hz. The lower panels show the instantaneous velocities calculated from the traces above. (E and F) Control experiments, under 8 pN of tension, showing that in the absence of GTP or EF-G, no translation signals were detected. https://doi.org/10.7554/eLife.03406.003 Ribosomes do not translate the message in a continuous manner, but in bursts of translation separated by long pauses (Figure 1B) that do not appear to be correlated with template position. As in all single-molecule experiments, there is a distribution of the noise level in the different single ribosome trajectories. As a result, some of the trajectories observed during translation bursts exhibit a particularly low noise level, and clearly show that the ribosome moves in periods of stationary dwells that are followed by translocation events corresponding to single codon steps (three nucleotides) along the mRNA (Figure 1, Figure 1—figure supplement 1). However, in most trajectories the noise prevents us from unambiguously identifying the individual steps, and we therefore base our analysis on the average properties of the translation bursts. We separate these pauses from active translation bursts using a velocity threshold (see 'Materials and methods'), and calculate the 'pause-free' velocity of the ribosome (Figure 1C). We find that as the opposing force, F, is increased, the mean pause-free velocity, v, decreases monotonically (Figure 2), indicating that mRNA translocation is rate limiting under the conditions of the experiment. Note that the applied force acts between the 3′ end of the mRNA and the small 30S subunit; there is no directly applied force between the mRNA and the large 50S subunit or between the 30S and the 50S subunits. In addition, the position of the attachment point (protein S16, in the 'back' of the 30S subunit) was chosen because it is remote of any known functional site and is not known to exhibit conformational dynamics during translocation. As a result, force only affects directly the mechanical step in which the anticodon loops of the tRNAs move from the A and P sites to the canonical P and E sites of the 30S subunit, respectively, together with the concomitant movement of the mRNA with respect to the ribosome. Hence, the position of the ribosome relative to the mRNA provides a convenient reaction coordinate to follow the translocation reaction during translational elongation. We can, thus, fit our data to an Arrhenius expression (1) v(F)=v0exp(−F·x˜kBT), where x˜ is the typical distance over which the force acts, v0 is the zero-force translocation velocity, kB is Boltzmann's constant and T = 296 K is the absolute temperature. The fit yields a zero-force velocity v0 = 2.9 codons/s (1.8, 4.0) and a distance x˜ = 1.4 nm (0.9, 1.8). The numbers in parenthesis indicate 95% confidence bounds. Figure 2 Download asset Open asset Pause-free translational velocity as a function of opposing force. Data points are the mean velocities for all measured traces at each force (N = 54). Error bars represent the standard error of the mean. The solid line is an exponential fit of the form v(F)=v0exp(−F·x˜kBT). https://doi.org/10.7554/eLife.03406.005 Notably, these results have implications for the intrinsic step size during translocation: crystal structures (Jenner et al., 2010) show that the distance between A- and P-site mRNA codons equals 1.48 nm. Clearly, at the end of translocation the mRNA has moved by this distance from its pre-translocation position; however, this value can either reflect a single, one-codon mechanical movement (a single barrier crossing in the free energy surface landscape) or successive smaller substeps (multiple barrier crossings) that sum to one codon, for example, three one-nucleotide substeps. Directly observing these potential substeps requires a temporal and spatial resolution that is not possible in our present experiment because the flexible nature of the mRNA and the relatively low forces involved give rise to high levels of thermally-induced fluctuations. However, because the distance x˜ determined here is similar to the measured one-codon translocation step, we can rule out one- and two-nucleotide translocation substeps and conclude that codon translocation is performed by the ribosome in a single step. In addition, our measurements shed light on the mechanism of translocation. Mechano-enzymes in general act by coupling a mechanical task (translocation, force generation, work) to a downhill chemical reaction (i.e., a reaction that lowers the total free energy of the system) (Bustamante et al., 2004). Clearly, given the diversity of conformational changes and chemical events associated with translocation by the ribosome, a complete description of the process should involve diffusion on a free-energy hypersurface with high dimensionality. However, given that our attachment geometry ensures that we affect and probe a single and well-defined mechanical coordinate, we can reduce this description to a simplified two-dimensional picture. In this two-dimensional free energy landscape one axis represents the mechanical coordinate that describes the movement of the mRNA relative to the 30S subunit, and the other axis the chemical coordinate that describes all binding, hydrolysis and dissociation processes (Figure 3) and, for the sake of simplicity, also conformational changes that have a reaction coordinate orthogonal to the reaction coordinate probed in our experiments. The most likely path for the reaction occurs along a minimum energy channel on this surface and the different events involved in translocation can now be described as diffusive transitions between minima of this (reduced) energy surface. Thus, for example, the classical-to-hybrid transitions and the associated ribosomal intersubunit rotations are assigned as movements along the chemical coordinate over a rather shallow activation energy that accounts for their reversible nature (Munro et al., 2007; Cornish et al., 2008; Fei et al., 2008). The three-dimensional energy surface depicted in Figure 3 naturally explains how transition rates are affected when a mechanical force is applied. The effect is equivalent to tilting the potential energy surface by rotating the diagram around its chemical axis (Bustamante et al., 2004), hence affecting the rate and equilibrium constants of reactions along the mechanical coordinate, for example making translocation more (force applied in the aiding or 'pushing' direction) or less (force applied in the opposing or 'pulling' direction) favorable. Figure 3 Download asset Open asset Reduced energy landscape for mRNA translocation. The mechanical coordinate describes the movement of the mRNA relative to the 30S subunit, while the chemical coordinate describes all binding, hydrolysis and dissociation processes, in addition to conformational changes with a reaction coordinate orthogonal to the translocation coordinate probed in our experiments. Translocation proceeds by diffusive transitions between minima of this reduced energy surface. A Power Stroke mechanism involves a diagonal transition, with simultaneous progress in the chemical and mechanical axis (dashed, purple line). Alternatively, a Brownian Ratchet (full, red lines) is composed of two orthogonal transitions: a fast equilibrium between pre- and post-translocated states along the mechanical coordinated, followed by a 'rectifying' chemical transition. https://doi.org/10.7554/eLife.03406.006 Translocation of mRNA and its two associated tRNA anticodon stem-loops from the A and P sites to the P and E sites of the 30S subunit (a movement along the 'mechanical' axis) must then be coupled to a downhill progress along the 'chemical' axis. Fundamentally, there are two ways in which this coupling can occur: one possibility is that the energy released by the 'chemical' transition is directly harnessed to produce the change. In this case, usually called a 'Power Stroke (PS)' mechanism, the system moves diagonally in the energy landscape. Alternatively, it is possible that the system moves back-and-forth spontaneously, driven by thermal energy, along the mechanical coordinate, until a chemical transition, that occurs when the system is in the post-translocated state, prevents the back-translocation and 'rectifies' this random motion into directed motion. This second mechanism, in which the system moves on the energy landscape in two orthogonal steps, is called a 'Brownian Ratchet (BR)'. Importantly, although both these different molecular mechanisms will result in a velocity which depends exponentially on the force, as in the Arrhenius equation above, they will differ in the identity of the distance x˜ (Wang et al., 1998): in the PS case, as the post-translocation state is achieved in a single (diagonal) transition, the force-dependence of the reaction rate will be given by an Arrhenius expression in which x˜=x††, that is, the distance along the mechanical coordinate to the transition state during translocation (Bustamante et al., 2004). Alternatively, in the BR case, the post-translocation state is achieved via two transitions, and the rate will be given by the product of the (force-dependent) probability of spontaneously populating the post-translocation state and the (force-independent) rate of the chemical reaction. As a result, the force dependence of the velocity is dictated by the force-dependent equilibrium constant between the pre- and post-translocated states, and hence described by an Arrhenius expression in which x˜=xstep, that is, equal to the distance between these states, or the step size of the motor (Bustamante et al., 2004). While these are two idealized cases and a real system is likely to combine features of these two models, the distance determined here (1.4 nm) is very close to the full 1.48 nm step size and indicates that, while we cannot fully rule out a PS mechanism, the ribosome likely functions as a BR during translocation. Notably, recent crystallographic studies of the ribosome bound to EF-G in a translocation intermediate (Chen et al., 2013; Pulk and Cate, 2013; Tourigny et al., 2013; Zhou et al., 2013), and a previous structure of the ribosome bound to EF-G in the post-translocation state (Gao et al., 2009) can lend support for this result and help clarify the identity of the reaction that rectifies translocation in this ratchet mechanism: the structures suggest that domain IV of EF-G could prevent back-translocation of the P-site tRNA by occupying the A-site, and that intercalation of two highly conserved bases of 16S rRNA into mRNA could prevent its back-translocation. These conformational changes could thus act as 'pawls' in the BR mechanism. Interestingly, the fact that both tRNA and mRNA movements would be locked could contribute to prevent frame shifting. The force at which the velocity approaches zero (the stall force) represents the maximum force that can be generated by the motor in a cycle. Our results indicate that the ribosome can forces as high as 2 the ribosome stall force is very close to that to unwind the structure present in mRNA et al., 2004). The between the mechanical of and the stall force of the ribosome indicates that RNA structures can have a effect on the rate of translation hence on such as frameshifting and of the and explains how these structures, while not can a regulatory role in the The work generated by the ribosome that is, the product of the stall force and the step pN nm = or about represents the mechanical work generated by the motor during the translocation step. is the for this mechanical The free-energy between peptide bond and bond hydrolysis is equivalent to 2 bond in our experimental conditions and Hence, the mechanical work that can be generated by the ribosome is of the total energy from and, in it is possible to translocation from this energy the to an from the hydrolysis of In studies have that the ribosome can in the absence of EF-G et al., or in the absence of GTP et al., and Noller, However, translocation is in many et al., 2006), and and translocation EF-G and GTP for of chemical energy to mechanical motion is than occurs in most molecular (Bustamante et al., 2004), and, it is not how the energy from peptide bond formation could be and from the 50S to the 30S in of our a mechanism in which EF-G binding and GTP hydrolysis for the energy of translocation and to be more the mechanism of of the ribosome as a during translocation is more than that of a typical molecular possible that from this and previous studies is that translocation is achieved by two during the first step, the ribosomal and relative to each other along an axis to the This reversible and is by the of the ends of the tRNAs in the classical A and P states into the hybrid A/P and P/E states and movement of the L1 of the tRNAs in their hybrid states and the rotation of the 30S subunit relative to the 50S Hence, binding of EF-G functions as a reaction for the first The ribosome acts as a for EF-G, and GTP hydrolysis conformational changes in the ribosome of the which opens the way for the of the P-site tRNA to the E that result in and transitions between the pre- and post-translocation This second BR is likely to be by the movement of domain IV of EF-G into the and the intercalation of two conserved bases of 16S rRNA into mRNA. then a of EF-G, the between and and the ribosome and in EF-G we our now to the pauses observed during translation. The of the translation bursts hence the is of the force opposing translocation (Figure the full distribution of is at all by a single exponential function with (Figure supplement 1). Figure shows that the of the pauses hence the exit is of force. the is also described by single exponential functions with the at all the forces (Figure supplement we conclude that into the state and from it are both by single, steps. Hence, we can all our measurements into one data and calculate the and exit = and = where the numbers in parenthesis indicate 95% confidence Figure with 1 supplement see all Download asset Open asset and exit (A) calculated as the of the mean of the translation bursts in between the pauses. exit equal to the of the mean rates are of the applied opposing force. is the of the observed It is known that ribosomes to at structure as hairpins and However, that were the for the observed their would be correlated with the position on the template we do not and their would depend on the applied force, as this force will the structure of the ribosome in the geometry used in these experiments. Thus, the force of the and exit rates out this Hence, we a in which these pauses represent events that are from the and translocation cycle. The rate limiting transitions into these states are steps along a reaction coordinate, or a mechanical coordinate that is orthogonal to the translocation movement, and hence not affected by the applied force. Interestingly, the and exit rates are very indicating that in the conditions of our the time by the ribosome in the translation states and the state are the The geometry of our a single reaction A more complete description of the translation process will a energy the assay here opens the way for by different attachment points on the the small and large for be possible to probe the of associated with ribosome translocation. Such should how this motor its dynamics with its translocation and Materials and methods of with biotinylated ribosomes a detailed A gene fusion encoding S16 linked via its C-terminus to the biotinylation domain of the biotin carboxyl carrier protein (BCCP) was introduced into the chromosome of coli, using the of et al. 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