X-Ray Analysis of Prokaryotic and Eukaryotic Ribosomes

Chapter
Part of the Biophysics for the Life Sciences book series (BIOPHYS, volume 1)

Abstract

The ribosome is the giant ribonucleoprotein assembly that translates the genetic code into protein in all living cells. Ribosomes from bacteria and archaea consist of a large (50S) and a small (30S) subunit, which together compose the 2.5 megadalton (MDa) 70S ribosome. Their eukaryotic counterparts are the 60S and 40S subunits and the 80S ribosome (from 3.5 MDa in lower eukaryotes to 4.5 MDa in higher). Many ribosomal key components are conserved across the three kingdoms of life: bacteria, archaea, and eukarya and constitutes a common core undertaking the fundamental processes of protein biosynthesis. The process of protein synthesis has been studied during the last five decades, and for most of this time, information of the three-dimensional structure of the ribosome has been vague and sparse. Cryo-electron microscopy and single-particle analysis produced the first direct visualizations of the bacterial ribosome in different functional states (Frank et al. 1995; Stark et al. 1997a, b; Agrawal et al. 1998). However, not until the X-ray crystallographic structures of the entire 70S ribosome as well as the individual 30S and 50S subunits began to emerge did accurate atomic models become available.

Over the last decade, remarkable advances have been made in the areas of ribosome crystallography, such that now it is possible to obtain at medium or high resolution not only the structure of the ribosome but also structures of the ribosome with key components bound such as messenger RNA (mRNA), transfer RNAs (tRNA), and various protein translation factors. This progress in elucidation of X-ray crystal structures concerned only bacterial and archaeal ribosomal studies because until 2010 no X-ray crystal structure of the eukaryote ribosome was available.

The overall goal of our research is to understand how the atomic structure of the ribosome ultimately determines its function. Towards this end we are using a combination of biochemical, biophysical, and molecular genetic methods directed at a model prokaryote (Thermus thermophilus) and eukaryote (the yeast Saccharomyces cerevisiae) organisms.

This chapter summarizes recent studies of the ribosome structures performed by X-ray crystallographic approaches, focusing primarily on recent work from our laboratory on prokaryotic 70S ribosomal complexes and on our recent progress in elucidation of first crystal structure of the complete 80S eukaryotic ribosome.

Keywords

Sugar Crystallization Magnesium Hydrated Codon 

References

  1. Agrawal RK, Penczek P, Grassucci RA, Frank J (1998) Visualization of elongation factor G on the Escherichia coli 70S ribosome: the mechanism of translocation. Proc Natl Acad Sci USA 95:6134–6138PubMedCrossRefGoogle Scholar
  2. Ashe MP, De Long SK, Sachs AB (2000) Glucose depletion rapidly inhibits translation initiation in yeast. Mol Biol Cell 11:833–848PubMedGoogle Scholar
  3. Ban N, Nissen P, Hansen J, Moore PB, Steitz TA (2000) The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. Science 289:905–920PubMedCrossRefGoogle Scholar
  4. Ben-Shem A, Jenner L, Yusupova G, Yusupov M (2010) Crystal structure of the eukaryotic ribosome. Science 330:1203–1209PubMedCrossRefGoogle Scholar
  5. Bouadloun F, Srichaiyo T, Isaksson LA, Bjork GR (1986) Influence of modification next to the anticodon in tRNA on codon context sensitivity of translational suppression and accuracy. J Bacteriol 166:1022–1027PubMedGoogle Scholar
  6. Bretscher MS (1968) Translocation in protein synthesis: a hybrid structure model. Nature 218:675–677PubMedCrossRefGoogle Scholar
  7. Brodersen DE, Clemons WM Jr, Carter AP, Wimberly BT, Ramakrishnan V (2002) Crystal structure of the 30S ribosomal subunit from Thermus thermophilus: structure of the proteins and their interactions with 16S RNA. J Mol Biol 316:725–768PubMedCrossRefGoogle Scholar
  8. Canonaco MA, Gualerzi CO, Pon CL (1989) Alternative occupancy of a dual ribosomal binding site by mRNA affected by translation initiation factors. Eur J Biochem 182:501–506PubMedCrossRefGoogle Scholar
  9. Dunkle JA, Wang L, Feldman MB, Pulk A, Chen VB et al (2011) Structures of the bacterial ­ribosome in classical and hybrid states of tRNA binding. Science 332:981–984PubMedCrossRefGoogle Scholar
  10. Frank J, Agrawal RK (2000) A ratchet-like inter-subunit reorganization of the ribosome during translocation. Nature 406:318–322PubMedCrossRefGoogle Scholar
  11. Frank J, Zhu J, Penczek P, Li Y, Srivastava S et al (1995) A model of protein synthesis based on cryo-electron microscopy of the E. coli ribosome. Nature 376:441–444PubMedCrossRefGoogle Scholar
  12. Gao H, Sengupta J, Valle M, Korostelev A, Eswar N et al (2003) Study of the structural dynamics of the E coli 70S ribosome using real-space refinement. Cell 113:789–801PubMedCrossRefGoogle Scholar
  13. Gustilo EM, Vendeix FA, Agris PF (2008) tRNA’s modifications bring order to gene expression. Curr Opin Microbiol 11:134–140PubMedCrossRefGoogle Scholar
  14. Halic M, Becker T, Frank J, Spahn CM, Beckmann R (2005) Localization and dynamic behavior of ribosomal protein L30e. Nat Struct Mol Biol 12:467–468PubMedCrossRefGoogle Scholar
  15. Harms J, Schluenzen F, Zarivach R, Bashan A, Gat S et al (2001) High resolution structure of the large ribosomal subunit from a mesophilic eubacterium. Cell 107:679–688PubMedCrossRefGoogle Scholar
  16. Hoburg A, Aschhoff HJ, Kersten H, Manderschied U, Gassen HG (1979) Function of modified nucleosides 7-methylguanosine, ribothymidine, and 2-thiomethyl-N6-(isopentenyl)adenosine in procaryotic transfer ribonucleic acid. J Bacteriol 140:408–414PubMedGoogle Scholar
  17. Horan LH, Noller HF (2007) Intersubunit movement is required for ribosomal translocation. Proc Natl Acad Sci USA 104:4881–4885PubMedCrossRefGoogle Scholar
  18. Jackson RJ (2005) Alternative mechanisms of initiating translation of mammalian mRNAs. Biochem Soc Trans 33:1231–1241PubMedCrossRefGoogle Scholar
  19. Jackson RJ, Hellen CU, Pestova TV (2010) The mechanism of eukaryotic translation initiation and principles of its regulation. Nat Rev Mol Cell Biol 11:113–127PubMedCrossRefGoogle Scholar
  20. Jenner L, Romby P, Rees B, Schulze-Briese C, Springer M et al (2005) Translational operator of mRNA on the ribosome: how repressor proteins exclude ribosome binding. Science 308:120–123PubMedCrossRefGoogle Scholar
  21. Jenner L, Rees B, Yusupov M, Yusupova G (2007) Messenger RNA conformations in the ribosomal E site revealed by X-ray crystallography. EMBO Rep 8:846–850PubMedCrossRefGoogle Scholar
  22. Jenner L, Demeshkina N, Yusupova G, Yusupov M (2010a) Structural rearrangements of the ribosome at the tRNA proofreading step. Nat Struct Mol Biol 17:1072–1078PubMedCrossRefGoogle Scholar
  23. Jenner LB, Demeshkina N, Yusupova G, Yusupov M (2010b) Structural aspects of messenger RNA reading frame maintenance by the ribosome. Nat Struct Mol Biol 17:555–560PubMedCrossRefGoogle Scholar
  24. Jorgensen F, Kurland CG (1990) Processivity errors of gene expression in Escherichia coli. J Mol Biol 215:511–521PubMedCrossRefGoogle Scholar
  25. Kaminishi T, Wilson DN, Takemoto C, Harms JM, Kawazoe M et al (2007) A snapshot of the 30S ribosomal subunit capturing mRNA via the Shine-Dalgarno interaction. Structure 15:289–297PubMedCrossRefGoogle Scholar
  26. Konevega AL, Soboleva NG, Makhno VI, Peshekhonov AV, Katunin VI (2006) The effect of modification of tRNA nucleotide-37 on the tRNA interaction with the P- and A-site of the 70S ribosome Escherichia coli. Mol Biol (Mosk) 40:669–683CrossRefGoogle Scholar
  27. Korostelev A, Trakhanov S, Laurberg M, Noller HF (2006) Crystal structure of a 70S ribosome-tRNA complex reveals functional interactions and rearrangements. Cell 126:1065–1077PubMedCrossRefGoogle Scholar
  28. Korostelev A, Trakhanov S, Asahara H, Laurberg M, Lancaster L et al (2007) Interactions and dynamics of the Shine Dalgarno helix in the 70S ribosome. Proc Natl Acad Sci USA 104:16840–16843PubMedCrossRefGoogle Scholar
  29. Korostelev A, Asahara H, Lancaster L, Laurberg M, Hirschi A et al (2008) Crystal structure of a translation termination complex formed with release factor RF2. Proc Natl Acad Sci USA 105:19684–19689PubMedCrossRefGoogle Scholar
  30. La Teana A, Gualerzi CO, Brimacombe R (1995) From stand-by to decoding site. Adjustment of the mRNA on the 30S ribosomal subunit under the influence of the initiation factors. RNA 1:772–782PubMedGoogle Scholar
  31. Laurberg M, Asahara H, Korostelev A, Zhu J, Trakhanov S et al (2008) Structural basis for translation termination on the 70S ribosome. Nature 454:852–857PubMedCrossRefGoogle Scholar
  32. Ma J, Campbell A, Karlin S (2002) Correlations between Shine-Dalgarno sequences and gene features such as predicted expression levels and operon structures. J Bacteriol 184:5733–5745PubMedCrossRefGoogle Scholar
  33. Menichi B, Heyman T (1976) Study of tyrosine transfer ribonucleic acid modification in relation to sporulation in Bacillus subtilis. J Bacteriol 127:268–280PubMedGoogle Scholar
  34. Ogle JM, Murphy FV, Tarry MJ, Ramakrishnan V (2002) Selection of tRNA by the ribosome requires a transition from an open to a closed form. Cell 111:721–732PubMedCrossRefGoogle Scholar
  35. Pisarev AV, Kolupaeva VG, Yusupov MM, Hellen CU, Pestova TV (2008) Ribosomal position and contacts of mRNA in eukaryotic translation initiation complexes. EMBO J 27:1609–1621PubMedCrossRefGoogle Scholar
  36. Rabl J, Leibundgut M, Ataide SF, Haag A, Ban N (2011) Crystal structure of the eukaryotic 40S ribosomal subunit in complex with initiation factor 1. Science 331:730–736PubMedCrossRefGoogle Scholar
  37. Rinke-Appel J, Junke N, Brimacombe R, Lavrik I, Dokudovskaya S et al (1994) Contacts between 16S ribosomal RNA and mRNA, within the spacer region separating the AUG initiator codon and the Shine-Dalgarno sequence; a site-directed cross-linking study. Nucleic Acids Res 22:3018–3025PubMedCrossRefGoogle Scholar
  38. Rozenski J, Crain PF, McCloskey JA (1999) The RNA modification database: 1999 update. Nucleic Acids Res 27:196–197PubMedCrossRefGoogle Scholar
  39. Schurr T, Nadir E, Margalit H (1993) Identification and characterization of E. coli ribosomal binding sites by free energy computation. Nucleic Acids Res 21:4019–4023PubMedCrossRefGoogle Scholar
  40. Schuwirth BS, Borovinskaya MA, Hau CW, Zhang W, Vila-Sanjurjo A et al (2005) Structures of the bacterial ribosome at 3.5 A resolution. Science 310:827–834PubMedCrossRefGoogle Scholar
  41. Selmer M, Dunham CM, Murphy FVt, Weixlbaumer A, Petry S et al (2006) Structure of the 70S ribosome complexed with mRNA and tRNA. Science 313:1935–1942PubMedCrossRefGoogle Scholar
  42. Sengupta J, Nilsson J, Gursky R, Spahn CM, Nissen P et al (2004) Identification of the versatile scaffold protein RACK1 on the eukaryotic ribosome by cryo-EM. Nat Struct Mol Biol 11:957–962PubMedCrossRefGoogle Scholar
  43. Shine J, Dalgarno L (1974) The 3′-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci USA 71:1342–1346PubMedCrossRefGoogle Scholar
  44. Sonenberg N, Hinnebusch AG (2009) Regulation of translation initiation in eukaryotes: mechanisms and biological targets. Cell 136:731–745PubMedCrossRefGoogle Scholar
  45. Spahn CM, Beckmann R, Eswar N, Penczek PA, Sali A et al (2001a) Structure of the 80S ribosome from Saccharomyces cerevisiae–tRNA-ribosome and subunit-subunit interactions. Cell 107:373–386PubMedCrossRefGoogle Scholar
  46. Spahn CM, Kieft JS, Grassucci RA, Penczek PA, Zhou K et al (2001b) Hepatitis C virus IRES RNA-induced changes in the conformation of the 40s ribosomal subunit. Science 291:1959–1962PubMedCrossRefGoogle Scholar
  47. Spahn CM, Gomez-Lorenzo MG, Grassucci RA, Jorgensen R, Andersen GR et al (2004a) Domain movements of elongation factor eEF2 and the eukaryotic 80S ribosome facilitate tRNA translocation. EMBO J 23:1008–1019PubMedCrossRefGoogle Scholar
  48. Spahn CM, Jan E, Mulder A, Grassucci RA, Sarnow P et al (2004b) Cryo-EM visualization of a viral internal ribosome entry site bound to human ribosomes: the IRES functions as an RNA-based translation factor. Cell 118:465–475PubMedCrossRefGoogle Scholar
  49. Spirin AS (1969) A model of the functioning ribosome: locking and unlocking of the ribosome subparticles. Cold Spring Harb Symp Quant Biol 34:197–207PubMedCrossRefGoogle Scholar
  50. Stark H, Orlova EV, Rinke-Appel J, Junke N, Mueller F et al (1997a) Arrangement of tRNAs in pre- and posttranslocational ribosomes revealed by electron cryomicroscopy. Cell 88:19–28PubMedCrossRefGoogle Scholar
  51. Stark H, Rodnina MV, Rinke-Appel J, Brimacombe R, Wintermeyer W et al (1997b) Visualization of elongation factor Tu on the Escherichia coli ribosome. Nature 389:403–406PubMedCrossRefGoogle Scholar
  52. Taylor DJ, Devkota B, Huang AD, Topf M, Narayanan E et al (2009) Comprehensive molecular structure of the eukaryotic ribosome. Structure 17:1591–1604PubMedCrossRefGoogle Scholar
  53. Urbonavicius J, Qian Q, Durand JM, Hagervall TG, Bjork GR (2001) Improvement of reading frame maintenance is a common function for several tRNA modifications. EMBO J 20:4863–4873PubMedCrossRefGoogle Scholar
  54. Urbonavicius J, Stahl G, Durand JM, Ben Salem SN, Qian Q et al (2003) Transfer RNA modifications that alter +1 frameshifting in general fail to affect −1 frameshifting. RNA 9:760–768PubMedCrossRefGoogle Scholar
  55. Valle M, Zavialov A, Sengupta J, Rawat U, Ehrenberg M et al (2003) Locking and unlocking of ribosomal motions. Cell 114:123–134PubMedCrossRefGoogle Scholar
  56. Weixlbaumer A, Murphy FV 4th, Dziergowska A, Malkiewicz A, Vendeix FA et al (2007a) Mechanism for expanding the decoding capacity of transfer RNAs by modification of uridines. Nat Struct Mol Biol 14:498–502PubMedCrossRefGoogle Scholar
  57. Weixlbaumer A, Petry S, Dunham CM, Selmer M, Kelley AC et al (2007b) Crystal structure of the ribosome recycling factor bound to the ribosome. Nat Struct Mol Biol 14:733–737PubMedCrossRefGoogle Scholar
  58. Wilson RK, Roe BA (1989) Presence of the hypermodified nucleotide N6-(delta 2-isopentenyl)-2-methylthioadenosine prevents codon misreading by Escherichia coli phenylalanyl-transfer RNA. Proc Natl Acad Sci USA 86:409–413PubMedCrossRefGoogle Scholar
  59. Wimberly BT, Brodersen DE, Clemons WM Jr, Morgan-Warren RJ, Carter AP et al (2000) Structure of the 30S ribosomal subunit. Nature 407:327–339PubMedCrossRefGoogle Scholar
  60. Yusupov MM, Yusupova GZ, Baucom A, Lieberman K, Earnest TN et al (2001) Crystal structure of the ribosome at 5.5 A resolution. Science 292:883–896PubMedCrossRefGoogle Scholar
  61. Yusupova GZ, Yusupov MM, Cate JH, Noller HF (2001) The path of messenger RNA through the ribosome. Cell 106:233–241PubMedCrossRefGoogle Scholar
  62. Yusupova G, Jenner L, Rees B, Moras D, Yusupov M (2006) Structural basis for messenger RNA movement on the ribosome. Nature 444:391–394PubMedCrossRefGoogle Scholar
  63. Zhang W, Dunkle JA, Cate JH (2009) Structures of the ribosome in intermediate states of ratcheting. Science 325:1014–1017PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  1. 1.Département de Biologie et de Génomique StructuralesInstitut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS, UMR7104, University of StrasbourgStrasbourg, IllkirchFrance

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