title: Direct measurement of the mechanical work during translocation by the ribosome.
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.
authors.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.
authors.forename: Tingting
authors.initials: T
authors.lastname: Liu
authors.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 Faculty of Biology, Technion-Israel Institute of Technology, Haifa, Israel Lorry I Lokey Interdisciplinary Center, Technion-Israel Institute of Technology, Haifa, Israel akaplanz@technion.ac.il.
authors.forename: Ariel
authors.initials: A
authors.lastname: Kaplan
authors.affiliation: Department of Chemistry, University of California, Berkeley, Berkeley, United States.
authors.forename: Lisa
authors.initials: L
authors.lastname: Alexander
authors.affiliation: Department of Chemistry, University of California, Berkeley, Berkeley, United States.
authors.forename: Shannon
authors.initials: S
authors.lastname: Yan
authors.affiliation: Department of Chemistry, University of California, Berkeley, Berkeley, United States.
authors.forename: Jin-Der
authors.initials: JD
authors.lastname: Wen
authors.affiliation: 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.
authors.forename: Laura
authors.initials: L
authors.lastname: Lancaster
authors.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.
authors.forename: Charles E
authors.initials: CE
authors.lastname: Wickersham
authors.forename: Kurt
authors.initials: K
authors.lastname: Fredrick
authors.affiliation: 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.
authors.forename: Kurt
authors.initials: K
authors.lastname: Fredrik
authors.affiliation: 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.
authors.forename: Harry
authors.initials: H
authors.lastname: Noller
authors.affiliation: Department of Chemistry, University of California, Berkeley, Berkeley, United States.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Tinoco
authors.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 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 akaplanz@technion.ac.il.
authors.forename: Carlos J
authors.initials: CJ
authors.lastname: Bustamante
mesh_terms: D001696:Biomechanical Phenomena; D003062:Codon / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli; D007700:Kinetics; D009038:Motion; D052898:Optical Tweezers; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry* / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D003062:Codon; D012333:RNA, Messenger; D012343:RNA, Transfer
keywords: mechanical force; optical tweezers; ribosome; single molecule; translation
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