Home LiteratureArticle Details
PMID: 20434456 Published · ppublish English Journal Article Research Support, N.I.H., Extramural

Conformational sampling of aminoacyl-tRNA during selection on the bacterial ribosome.

Journal of molecular biology ·Vol. 399 ·No. 4 ·2010-06-18 ·Pages 576-95

Geggier P, Dave R, Feldman MB, Terry DS, Altman RB, Munro JB, Blanchard SC

Abstract

Aminoacyl-tRNA (aa-tRNA), in a ternary complex with elongation factor-Tu and GTP, enters the aminoacyl (A) site of the ribosome via a multi-step, mRNA codon-dependent mechanism. This process gives rise to the preferential selection of cognate aa-tRNAs for each mRNA codon and, consequently, the fidelity of gene expression. The ribosome actively facilitates this process by recognizing structural features of the correct substrate, initiated in its decoding site, to accelerate the rates of elongation factor-Tu-catalyzed GTP hydrolysis and ribosome-catalyzed peptide bond formation. Here, the order and timing of conformational events underpinning the aa-tRNA selection process were investigated from multiple structural perspectives using single-molecule fluorescence resonance energy transfer. The time resolution of these measurements was extended to 2.5 and 10 ms, a 10- to 50-fold improvement over previous studies. The data obtained reveal that aa-tRNA undergoes fast conformational sampling within the A site, both before and after GTP hydrolysis. This suggests that the alignment of aa-tRNA with respect to structural elements required for irreversible GTP hydrolysis and peptide bond formation plays a key role in the fidelity mechanism. These observations provide direct evidence that the selection process is governed by motions of aa-tRNA within the A site, adding new insights into the physical framework that helps explain how the rates of GTP hydrolysis and peptide bond formation are controlled by the mRNA codon and other fidelity determinants within the system.

MeSH Terms
Codon/genetics,metabolism Escherichia coli/genetics,metabolism Fluorescence Resonance Energy Transfer Fluorescent Dyes Guanosine Triphosphate/chemistry,metabolism Hydrolysis Kinetics Macromolecular Substances Models, Biological Models, Molecular Nucleic Acid Conformation Peptide Elongation Factor Tu/chemistry,metabolism RNA, Bacterial/chemistry,genetics,metabolism RNA, Transfer, Amino Acyl/chemistry,metabolism RNA, Transfer, Met/chemistry,metabolism RNA, Transfer, Phe/chemistry,metabolism Ribosomes/metabolism
Chemicals
Codon Fluorescent Dyes Macromolecular Substances RNA, Bacterial RNA, Transfer, Amino Acyl RNA, Transfer, Met RNA, Transfer, Phe tRNA, formylmethionine- Guanosine Triphosphate Peptide Elongation Factor Tu
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
Geggier Peter
Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University, 1300 York Avenue, New York, NY 10065, USA.
Dave Richa
Feldman Michael B
Terry Daniel S
Altman Roger B
Munro James B
Blanchard Scott C
References (59)
59 references, click to expand
  1. Ribosome fidelity: tRNA discrimination, proofreading and induced fit.
    Trends Biochem Sci. 2001 Feb;26(2):124-30 PMID: 11166571
  2. Structural insights into translational fidelity.
    Annu Rev Biochem. 2005;74:129-77 PMID: 15952884
  3. tRNA dynamics on the ribosome during translation.
    Proc Natl Acad Sci U S A. 2004 Aug 31;101(35):12893-8 PMID: 15317937
  4. GTPase activation of elongation factor EF-Tu by the ribosome during decoding.
    EMBO J. 2009 Mar 18;28(6):755-65 PMID: 19229291
  5. Spontaneous formation of the unlocked state of the ribosome is a multistep process.
    Proc Natl Acad Sci U S A. 2010 Jan 12;107(2):709-14 PMID: 20018653
  6. The crystal structure of the ribosome bound to EF-Tu and aminoacyl-tRNA.
    Science. 2009 Oct 30;326(5953):688-694 PMID: 19833920
  7. Transfer RNA structure and coding specificity. I. Evidence that a D-arm mutation reduces tRNA dissociation from the ribosome.
    J Mol Biol. 1989 Apr 5;206(3):489-501 PMID: 2469803
  8. GTPase activation of elongation factors Tu and G on the ribosome.
    Biochemistry. 2002 Oct 15;41(41):12520-8 PMID: 12369843
  9. Fidelity of aminoacyl-tRNA selection on the ribosome: kinetic and structural mechanisms.
    Annu Rev Biochem. 2001;70:415-35 PMID: 11395413
  10. Kinetic determinants of high-fidelity tRNA discrimination on the ribosome.
    Mol Cell. 2004 Jan 30;13(2):191-200 PMID: 14759365
  11. Fluorescence spectroscopy of single biomolecules.
    Science. 1999 Mar 12;283(5408):1676-83 PMID: 10073925
  12. Characterization of 16S rRNA mutations that decrease the fidelity of translation initiation.
    RNA. 2007 Dec;13(12):2348-55 PMID: 17942743
  13. Ribosome-induced changes in elongation factor Tu conformation control GTP hydrolysis.
    Proc Natl Acad Sci U S A. 2009 Jan 27;106(4):1063-8 PMID: 19122150
  14. Mitigating unwanted photophysical processes for improved single-molecule fluorescence imaging.
    Biophys J. 2009 Mar 18;96(6):2371-81 PMID: 19289062
  15. Costs of accuracy determined by a maximal growth rate constraint.
    Q Rev Biophys. 1984 Feb;17(1):45-82 PMID: 6484121
  16. The ribosome's response to codon-anticodon mismatches.
    Biochimie. 2006 Aug;88(8):1001-11 PMID: 16716484
  17. RNA CODEWORDS AND PROTEIN SYNTHESIS. THE EFFECT OF TRINUCLEOTIDES UPON THE BINDING OF SRNA TO RIBOSOMES.
    Science. 1964 Sep 25;145(3639):1399-407 PMID: 14172630
  18. Allosteric mechanism for codon-dependent tRNA selection on ribosomes.
    Proc Natl Acad Sci U S A. 1975 Nov;72(11):4248-51 PMID: 1060103
  19. Alignment/misalignment hypothesis for tRNA selection by the ribosome.
    Biochimie. 2006 Aug;88(8):1075-89 PMID: 16890341
  20. Ribosome structure and the mechanism of translation.
    Cell. 2002 Feb 22;108(4):557-72 PMID: 11909526
  21. How RNA unfolds and refolds.
    Annu Rev Biochem. 2008;77:77-100 PMID: 18518818
  22. Simulating movement of tRNA into the ribosome during decoding.
    Proc Natl Acad Sci U S A. 2005 Nov 1;102(44):15854-9 PMID: 16249344
  23. Kinetic amplification of enzyme discrimination.
    Biochimie. 1975;57(5):587-95 PMID: 1182215
  24. What recent ribosome structures have revealed about the mechanism of translation.
    Nature. 2009 Oct 29;461(7268):1234-42 PMID: 19838167
  25. Hasty decisions on the ribosome.
    Nat Struct Mol Biol. 2004 Mar;11(3):206-8 PMID: 14983168
  26. Visualization of elongation factor Tu on the Escherichia coli ribosome.
    Nature. 1997 Sep 25;389(6649):403-6 PMID: 9311785
  27. Exploration of the transition state for tertiary structure formation between an RNA helix and a large structured RNA.
    J Mol Biol. 2003 May 16;328(5):1011-26 PMID: 12729738
  28. Recognition of aminoacyl-tRNA: a common molecular mechanism revealed by cryo-EM.
    EMBO J. 2008 Dec 17;27(24):3322-31 PMID: 19020518
  29. Transient conformational states of aminoacyl-tRNA during ribosome binding catalyzed by elongation factor Tu.
    Biochemistry. 1994 Oct 11;33(40):12267-75 PMID: 7918447
  30. The reaction of ribosomes with elongation factor Tu.GTP complexes. Aminoacyl-tRNA-independent reactions in the elongation cycle determine the accuracy of protein synthesis.
    J Biol Chem. 1986 Apr 15;261(11):4868-74 PMID: 3514605
  31. Rate and accuracy of bacterial protein synthesis revisited.
    Curr Opin Microbiol. 2008 Apr;11(2):141-7 PMID: 18400551
  32. Restoration of single-channel currents using the segmental k-means method based on hidden Markov modeling.
    Biophys J. 2004 Mar;86(3):1488-501 PMID: 14990476
  33. Is there proofreading during polypeptide synthesis?
    EMBO J. 1982;1(6):741-5 PMID: 6765234
  34. Initial binding of the elongation factor Tu.GTP.aminoacyl-tRNA complex preceding codon recognition on the ribosome.
    J Biol Chem. 1996 Jan 12;271(2):646-52 PMID: 8557669
  35. Analysis of single-molecule FRET trajectories using hidden Markov modeling.
    Biophys J. 2006 Sep 1;91(5):1941-51 PMID: 16766620
  36. The role of tRNA as a molecular spring in decoding, accommodation, and peptidyl transfer.
    FEBS Lett. 2005 Feb 7;579(4):959-62 PMID: 15680982
  37. Rate, accuracy and cost of ribosomes in bacterial cells.
    Biochimie. 2006 Aug;88(8):951-61 PMID: 16890342
  38. tRNA selection and kinetic proofreading in translation.
    Nat Struct Mol Biol. 2004 Oct;11(10):1008-14 PMID: 15448679
  39. Induced fit in initial selection and proofreading of aminoacyl-tRNA on the ribosome.
    EMBO J. 1999 Jul 1;18(13):3800-7 PMID: 10393195
  40. Proofreading of the codon-anticodon interaction on ribosomes.
    Proc Natl Acad Sci U S A. 1977 Jan;74(1):198-202 PMID: 319457
  41. Kinetic proofreading: a new mechanism for reducing errors in biosynthetic processes requiring high specificity.
    Proc Natl Acad Sci U S A. 1974 Oct;71(10):4135-9 PMID: 4530290
  42. Fluctuations of transfer RNAs between classical and hybrid states.
    Biophys J. 2007 Nov 15;93(10):3575-82 PMID: 17693476
  43. The role of fluctuations in tRNA selection by the ribosome.
    Proc Natl Acad Sci U S A. 2007 Aug 21;104(34):13661-5 PMID: 17699629
  44. Cryo-EM reveals an active role for aminoacyl-tRNA in the accommodation process.
    EMBO J. 2002 Jul 1;21(13):3557-67 PMID: 12093756
  45. Long-range signalling in activation of the translational GTPase EF-Tu.
    EMBO J. 2009 Mar 18;28(6):619-20 PMID: 19295500
  46. An active role for tRNA in decoding beyond codon:anticodon pairing.
    Science. 2005 May 20;308(5725):1178-80 PMID: 15905403
  47. A uniform response to mismatches in codon-anticodon complexes ensures ribosomal fidelity.
    Mol Cell. 2006 Feb 3;21(3):369-77 PMID: 16455492
  48. Complete kinetic mechanism of elongation factor Tu-dependent binding of aminoacyl-tRNA to the A site of the E. coli ribosome.
    EMBO J. 1998 Dec 15;17(24):7490-7 PMID: 9857203
  49. Recognition and selection of tRNA in translation.
    FEBS Lett. 2005 Feb 7;579(4):938-42 PMID: 15680978
  50. Errors and alternatives in reading the universal genetic code.
    Microbiol Rev. 1989 Sep;53(3):273-98 PMID: 2677635
  51. Identification of two distinct hybrid state intermediates on the ribosome.
    Mol Cell. 2007 Feb 23;25(4):505-17 PMID: 17317624
  52. Structural basis for the function of the ribosomal L7/12 stalk in factor binding and GTPase activation.
    Cell. 2005 Jul 1;121(7):991-1004 PMID: 15989950
  53. Energetic contribution of tRNA hybrid state formation to translocation catalysis on the ribosome.
    Nat Struct Biol. 2000 Nov;7(11):1027-31 PMID: 11062557
  54. Insights into the decoding mechanism from recent ribosome structures.
    Trends Biochem Sci. 2003 May;28(5):259-66 PMID: 12765838
  55. Incorporation of aminoacyl-tRNA into the ribosome as seen by cryo-electron microscopy.
    Nat Struct Biol. 2003 Nov;10(11):899-906 PMID: 14566331
  56. Accommodation of aminoacyl-tRNA into the ribosome involves reversible excursions along multiple pathways.
    RNA. 2010 Jun;16(6):1196-204 PMID: 20427512
  57. Translation at the single-molecule level.
    Annu Rev Biochem. 2008;77:177-203 PMID: 18518820
  58. Crystal structure of the ribosome at 5.5 A resolution.
    Science. 2001 May 4;292(5518):883-96 PMID: 11283358
  59. Multiple stages in codon-anticodon recognition: double-trigger mechanisms and geometric constraints.
    Biochimie. 2006 Aug;88(8):963-92 PMID: 16843583
Article Info
Journal
Journal of molecular biology
Abbr.
J Mol Biol
ISSN
1089-8638
DOI
10.1016/j.jmb.2010.04.038
Published
2010-06-18
Epub
2010-00-29
Pages
576-95
Language
English
Region
England
NLM ID
2985088R
PMCID
PMC2917329
Subset
IM
Grants
NIGMS NIH HHS · R01 GM079238 · United States
NIGMS NIH HHS · R01 GM079238-04 · United States
NIGMS NIH HHS · T32 GM007739 · United States
NIGMS NIH HHS · GM079238 · United States
External Links
PubMed source DOI: 10.1016/j.jmb.2010.04.038 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: [email protected]

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2