Direct measurement of the mechanical work during translocation by the ribosome.
Bottom Line: 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.
Affiliation: Jason L Choy Laboratory of Single Molecule Biophysics, University of California, Berkeley, Berkeley, United States Department of Physics, University of California, Berkeley, Berkeley, United States.Show MeSH
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Mentions: 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.10.7554/eLife.03406.006Figure 3.Reduced energy landscape for mRNA translocation.
Affiliation: Jason L Choy Laboratory of Single Molecule Biophysics, University of California, Berkeley, Berkeley, United States Department of Physics, University of California, Berkeley, Berkeley, United States.