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      Real-time tRNA transit on single translating ribosomes at codon resolution

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          Abstract

          Translation by the ribosome occurs by a complex mechanism involving the coordinated interaction of multiple nucleic acid and protein ligands. Here we have used zero-mode waveguides (ZMWs) and sophisticated detection instrumentation to allow real-time observation of translation at physiologically-relevant (μM) ligand concentrations. Translation at each codon is monitored by stable binding of tRNAs – labeled with distinct fluorophores – to translating ribosomes, allowing direct detection of the identity of tRNA molecules bound to the ribosome, and therefore, the underlying mRNA sequence. We observe the transit of tRNAs on single translating ribosomes and have determined the number of tRNA molecules simultaneously bound to the ribosome, at each codon of an mRNA. Our results show that ribosomes are only briefly occupied by two tRNAs and that release of deacylated tRNA from the E site is uncoupled from binding of A-site tRNA and occurs rapidly after translocation. The methods outlined here have broad application to the study of mRNA sequences, and the mechanism and regulation of translation.

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          Most cited references24

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          Zero-mode waveguides for single-molecule analysis at high concentrations.

          Optical approaches for observing the dynamics of single molecules have required pico- to nanomolar concentrations of fluorophore in order to isolate individual molecules. However, many biologically relevant processes occur at micromolar ligand concentrations, necessitating a reduction in the conventional observation volume by three orders of magnitude. We show that arrays of zero-mode waveguides consisting of subwavelength holes in a metal film provide a simple and highly parallel means for studying single-molecule dynamics at micromolar concentrations with microsecond temporal resolution. We present observations of DNA polymerase activity as an example of the effectiveness of zero-mode waveguides for performing single-molecule experiments at high concentrations.
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            Intermediate states in the movement of transfer RNA in the ribosome.

            Direct chemical 'footprinting' shows that translocation of transfer RNA occurs in two discrete steps. During the first step, which occurs spontaneously after the formation of the peptide bond, the acceptor end of tRNA moves relative to the large ribosomal subunit resulting in 'hybrid states' of binding. During the second step, which is promoted by elongation factor EF-G, the anticodon end of tRNA, along with the messenger RNA, moves relative to the small ribosomal subunit.
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              tRNA selection and kinetic proofreading in translation.

              Using single-molecule methods we observed the stepwise movement of aminoacyl-tRNA (aa-tRNA) into the ribosome during selection and kinetic proofreading using single-molecule fluorescence resonance energy transfer (smFRET). Intermediate states in the pathway of tRNA delivery were observed using antibiotics and nonhydrolyzable GTP analogs. We identified three unambiguous FRET states corresponding to initial codon recognition, GTPase-activated and fully accommodated states. The antibiotic tetracycline blocks progression of aa-tRNA from the initial codon recognition state, whereas cleavage of the sarcin-ricin loop impedes progression from the GTPase-activated state. Our data support a model in which ribosomal recognition of correct codon-anticodon pairs drives rotational movement of the incoming complex of EF-Tu-GTP-aa-tRNA toward peptidyl-tRNA during selection on the ribosome. We propose a mechanistic model of initial selection and proofreading.
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                Author and article information

                Journal
                0410462
                6011
                Nature
                Nature
                Nature
                0028-0836
                1476-4687
                9 June 2015
                15 April 2010
                15 June 2015
                : 464
                : 7291
                : 1012-1017
                Affiliations
                [1 ] Department of Structural Biology, Stanford University School of Medicine, Stanford, California USA 94305-5126
                [2 ] Japan Science and Technology Agency, 4-1-8, Honcho, Kawaguchi, Saitama 332-0012, Japan.
                [3 ] Biophysics Program, Stanford University, 1505 Adams Drive, Menlo Park, California 94025
                [4 ] Pacific Biosciences, Inc., 1505 Adams Drive, Menlo Park, California 94025
                Author notes
                Correspondence and requests for materials should be addressed to J.D.P. ( puglisi@ 123456stanford.edu ).
                Article
                NIHMS696686
                10.1038/nature08925
                4466108
                20393556
                59248038-c6c2-4781-a76a-e050de1ad1b2

                Reprints and permissions information is available at www.nature.com/reprints.

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