title: EF-G catalyzes tRNA translocation by disrupting interactions between decoding center and codon-anticodon duplex.
abstract: During translation, elongation factor G (EF-G) catalyzes the translocation of tRNA2-mRNA inside the ribosome. Translocation is coupled to a cycle of conformational rearrangements of the ribosomal machinery, and how EF-G initiates translocation remains unresolved. Here we performed systematic mutagenesis of Escherichia coli EF-G and analyzed inhibitory single-site mutants of EF-G that preserved pretranslocation (Pre)-state ribosomes with tRNAs in A/P and P/E sites (Pre-EF-G). Our results suggest that the interactions between the decoding center and the codon-anticodon duplex constitute the barrier for translocation. Catalysis of translocation by EF-G involves the factor's highly conserved loops I and II at the tip of domain IV, which disrupt the hydrogen bonds between the decoding center and the duplex to release the latter, hence inducing subsequent translocation events, namely 30S head swiveling and tRNA2-mRNA movement on the 30S subunit.
authors.affiliation: Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Guangqiao
authors.initials: G
authors.lastname: Liu
authors.affiliation: Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Guangtao
authors.initials: G
authors.lastname: Song
authors.affiliation: Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Danyang
authors.initials: D
authors.lastname: Zhang
authors.affiliation: Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Dejiu
authors.initials: D
authors.lastname: Zhang
authors.affiliation: Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Zhikai
authors.initials: Z
authors.lastname: Li
authors.affiliation: Beijing National Laboratory for Molecular Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Department of Chemical Biology, College of Chemistry and Molecular Engineering, and Biodynamic Optical Imaging Center, Peking University, Beijing, China.
authors.forename: Zhixin
authors.initials: Z
authors.lastname: Lyu
authors.affiliation: Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Jianshu
authors.initials: J
authors.lastname: Dong
authors.affiliation: NOXXON Pharma AG, Berlin, Germany.
authors.forename: John
authors.initials: J
authors.lastname: Achenbach
authors.affiliation: Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Weimin
authors.initials: W
authors.lastname: Gong
authors.affiliation: Beijing National Laboratory for Molecular Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Department of Chemical Biology, College of Chemistry and Molecular Engineering, and Biodynamic Optical Imaging Center, Peking University, Beijing, China.
authors.forename: Xin Sheng
authors.initials: XS
authors.lastname: Zhao
authors.affiliation: Charité, Institute for Medical Physics and Biophysics, Berlin, Germany.
authors.forename: Knud H
authors.initials: KH
authors.lastname: Nierhaus
authors.affiliation: Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Qin
mesh_terms: D000595:Amino Acid Sequence; D000926:Anticodon / Q000378:metabolism*; D003062:Codon / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D020653:Peptide Elongation Factor G / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D034443:RNA Transport; D012343:RNA, Transfer / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D000926:Anticodon; D003062:Codon; D029968:Escherichia coli Proteins; D020653:Peptide Elongation Factor G; D012343:RNA, Transfer
references.citation: RNA. 2005 Nov;11(11):1624-32
references.pmid: 16177132
references.citation: Nat Rev Microbiol. 2014 Feb;12(2):89-100
references.pmid: 24362468
references.citation: J Mol Biol. 2005 Nov 25;354(2):459-72
references.pmid: 16243353
references.citation: Proc Natl Acad Sci U S A. 2007 Dec 11;104(50):19671-8
references.pmid: 18003906
references.citation: J Mol Biol. 2010 Mar 5;396(4):1043-52
references.pmid: 20045415
references.citation: Biochemistry. 2002 Oct 22;41(42):12806-12
references.pmid: 12379123
references.citation: J Mol Biol. 2000 Jul 21;300(4):951-61
references.pmid: 10891280
references.citation: Proc Natl Acad Sci U S A. 2012 Oct 16;109(42):16900-5
references.pmid: 23027944
references.citation: Nature. 1997 Jan 2;385(6611):37-41
references.pmid: 8985244
references.citation: Trends Biochem Sci. 2009 Aug;34(8):390-400
references.pmid: 19647434
references.citation: Science. 2013 Jun 28;340(6140):1235970
references.pmid: 23812721
references.citation: Cell. 2006 Nov 17;127(4):721-33
references.pmid: 17110332
references.citation: Proteins. 2011 May;79(5):1478-86
references.pmid: 21365677
references.citation: Mol Cell. 2001 May;7(5):1037-45
references.pmid: 11389850
references.citation: Biochem J. 2013 Jun 1;452(2):173-81
references.pmid: 23662805
references.citation: Mol Cell. 2011 May 6;42(3):367-77
references.pmid: 21549313
references.citation: Nat Commun. 2013;4:1477
references.pmid: 23403578
references.citation: EMBO J. 2011 Apr 20;30(8):1497-507
references.pmid: 21378755
references.citation: J Biol Chem. 2009 Aug 7;284(32):21103-19
references.pmid: 19416977
references.citation: Nat Struct Mol Biol. 2011 Oct 23;18(11):1300-2
references.pmid: 22020300
references.citation: Nature. 2012 Mar 21;484(7393):256-9
references.pmid: 22437501
references.citation: J Mol Biol. 2005 May 13;348(4):939-49
references.pmid: 15843024
references.citation: Science. 2009 Aug 21;325(5943):1014-7
references.pmid: 19696352
references.citation: PLoS One. 2012;7(9):e46068
references.pmid: 23049938
references.citation: Protein Expr Purif. 2000 Apr;18(3):257-61
references.pmid: 10733877
references.citation: Methods Enzymol. 1997;276:307-26
references.pmid: 27754618
references.citation: Annu Rev Physiol. 1993;55:397-426
references.pmid: 8466181
references.citation: Mol Cell. 2002 Oct;10(4):799-807
references.pmid: 12419224
references.citation: Proc Natl Acad Sci U S A. 2013 Dec 24;110(52):20964-9
references.pmid: 24324168
references.citation: Nat Struct Mol Biol. 2008 Sep;15(9):910-5
references.pmid: 19172743
references.citation: Mol Cell. 2008 Oct 24;32(2):292-9
references.pmid: 18951096
references.citation: Cell. 2002 Nov 27;111(5):721-32
references.pmid: 12464183
references.citation: Nat Struct Mol Biol. 2013 Sep;20(9):1077-84
references.pmid: 23912278
references.citation: Cell. 2004 May 28;117(5):589-99
references.pmid: 15163407
references.citation: Curr Opin Struct Biol. 2012 Dec;22(6):778-85
references.pmid: 22906732
references.citation: RNA. 2001 Dec;7(12):1879-85
references.pmid: 11780642
references.citation: EMBO J. 1994 Aug 15;13(16):3661-8
references.pmid: 8070396
references.citation: J Mol Biol. 2013 Oct 23;425(20):3778-89
references.pmid: 23542008
references.citation: Proc Natl Acad Sci U S A. 2013 Dec 24;110(52):20994-9
references.pmid: 24324137
references.citation: EMBO J. 1998 Jun 15;17(12):3478-83
references.pmid: 9628883
references.citation: Nat Struct Mol Biol. 2012 Mar 11;19(4):403-10
references.pmid: 22407015
references.citation: FEBS Lett. 1999 Jun 11;452(3):155-9
references.pmid: 10386581
references.citation: Mol Cell. 2009 Aug 28;35(4):502-10
references.pmid: 19716793
references.citation: Proc Natl Acad Sci U S A. 1996 Apr 30;93(9):4202-6
references.pmid: 8633041
references.citation: Cold Spring Harb Symp Quant Biol. 2009;74:25-33
references.pmid: 19955257
references.citation: Cold Spring Harb Perspect Biol. 2011 Nov 01;3(11):a003780
references.pmid: 21068149
references.citation: Proc Natl Acad Sci U S A. 2012 Dec 11;109(50):20391-4
references.pmid: 23188795
references.citation: PLoS One. 2013;8(3):e58829
references.pmid: 23527033
references.citation: Science. 2013 Jun 28;340(6140):1235490
references.pmid: 23812720
references.citation: Science. 2009 Oct 30;326(5953):694-9
references.pmid: 19833919
references.citation: EMBO J. 2003 Feb 17;22(4):945-53
references.pmid: 12574130
references.citation: J Mol Biol. 1977 Sep 25;115(3):335-54
references.pmid: 338910
references.citation: RNA. 2007 Sep;13(9):1473-82
references.pmid: 17630323
references.citation: Science. 2001 May 4;292(5518):897-902
references.pmid: 11340196
references.citation: Methods Enzymol. 2000;318:446-60
references.pmid: 10890005
references.citation: Science. 2013 Jun 28;340(6140):1236086
references.pmid: 23812722
references.citation: Nucleic Acids Res. 2012 Nov;40(21):10851-65
references.pmid: 22965132
references.citation: Proc Natl Acad Sci U S A. 2011 Sep 27;108(39):16223-8
references.pmid: 21930951
references.citation: J Mol Biol. 2000 Nov 3;303(4):593-603
references.pmid: 11054294
references.citation: Nature. 2010 Dec 2;468(7324):713-6
references.pmid: 21124459
references.citation: Proc Natl Acad Sci U S A. 2008 Jul 8;105(27):9192-7
references.pmid: 18591673
references.citation: J Mol Biol. 1990 Jan 5;211(1):135-45
references.pmid: 2405162
references.citation: Nature. 2000 Jul 20;406(6793):318-22
references.pmid: 10917535