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 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).10.7554/eLife.03406.003Figure 1.Following translation by a single ribosome on a single mRNA.
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.