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
Mentions: 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 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 = 1.4 nm (0.9, 1.8). The numbers in parenthesis indicate 95% confidence bounds.10.7554/eLife.03406.005Figure 2.Pause-free translational velocity as a function of opposing force.
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.