Direct measurement of the mechanical work during translocation by the ribosome
Tingting Liu,
Ariel Kaplan,
Lisa Alexander,
Shannon Yan,
Jin-Der Wen,
Laura Lancaster,
Charles E Wickersham,
Kurt Fredrick,
Harry Noller,
Ignacio Tinoco Jr,
Carlos J Bustamante
Affiliations
Tingting Liu
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
Ariel Kaplan
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; Faculty of Biology, Technion-Israel Institute of Technology, Haifa, Israel; Lorry I Lokey Interdisciplinary Center, Technion-Israel Institute of Technology, Haifa, Israel
Lisa Alexander
Department of Chemistry, University of California, Berkeley, Berkeley, United States
Shannon Yan
Department of Chemistry, University of California, Berkeley, Berkeley, United States
Jin-Der Wen
Department of Chemistry, University of California, Berkeley, Berkeley, United States
Laura Lancaster
Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, Santa Cruz, United States; Center for Molecular Biology of RNA, University of California, Santa Cruz, Santa Cruz, United States
Charles E Wickersham
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
Kurt Fredrick
Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, Santa Cruz, United States; Center for Molecular Biology of RNA, University of California, Santa Cruz, Santa Cruz, United States
Harry Noller
Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, Santa Cruz, United States; Center for Molecular Biology of RNA, University of California, Santa Cruz, Santa Cruz, United States
Ignacio Tinoco Jr
Department of Chemistry, University of California, Berkeley, Berkeley, United States
Carlos J Bustamante
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; Department of Chemistry, University of California, Berkeley, Berkeley, United States; California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, United States; Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States
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.
