Skip to main content
SpringerLink
Log in
Book cover

Ribosomes pp 393–404Cite as

Nascent polypeptide chains within the ribosomal tunnel analyzed by cryo-EM

  • Chapter

  • 2252 Accesses

Abstract

The ribosome is a large macromolecular particle that synthesizes polypeptide chains from the substituent amino acid building blocks. The active site for peptide bond formation, the so-called peptidyl transferase center (PTC), is located in a cleft on the intersubunit side of the large ribosomal subunit (reviewed by (Polacek and Mankin, 2005; Simonovic and Steitz, 2009)). As the nascent polypeptide chain is being synthesized, it passes through a tunnel within the large subunit and emerges at the solvent side where protein folding occurs. The first hints for the presence of a ribosomal tunnel in the large subunit came from proteolysis protection and immuno-electron microscopy (EM) studies: Using IgG antibodies raised against β-galactosidase or the rubisco small subunit, Lake and coworkers could show that polypeptide chains emerge on the back of large subunit of the bacterial (Escherichia coli) 70S and eukaryotic (plant) 80S ribosome, respectively — some 75 Å from the intersubunit interface (Bernabeu and Lake, 1982; Bernabeu et al., 1983). This distance was consistent with the earlier findings that 30–40 C-terminal amino acids of nascent polypeptide chains are protected by eukaryotic and bacterial ribosomes from proteolysis (Malkin and Rich, 1967; Blobel and Sabatini, 1970; Smith et al., 1978).

This is a preview of subscription content, log in via an institution.

Chapter
USD   29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   169.00
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   219.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  • Arai R, Ueda H, Kitayama A, Kamiya N, Nagamune T (2001) Design of the linkers which effectively separate domains of a bifunctional fusion protein. Protein Eng 14: 529–532

    Article  PubMed  CAS  Google Scholar 

  • Ban N, Nissen P, Hansen J, Moore PB, Steitz TA (2000) The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution. Science 289: 905–920

    Article  PubMed  CAS  Google Scholar 

  • Bashan A, Agmon I, Zarivach R, Schluenzen F, Harms J, Berisio R, Bartels H, Franceschi F, Auerbach T, Hansen HA, Kossoy E, Kessler M, Yonath A (2003) Structural basis of the ribosomal machinery for peptide bond formation, translocation, and nascent chain progression. Mol Cell 11: 91–102

    Article  PubMed  CAS  Google Scholar 

  • Becker T, Bhushan S, Jarasch A, Armache JP, Funes S, Jossinet F, Gumbart J, Mielke T, Berninghausen O, Schulten K, Westhof E, Gilmore R, Mandon EC, Beckmann R (2009) Structure of monomeric yeast and mammalian Sec61 complexes interacting with the translating ribosome. Science 326: 1369–1373

    Article  PubMed  CAS  Google Scholar 

  • Beckmann R, Bubeck D, Grassucci R, Penczek P, Verschoor A, Blobel G, Frank J (1997) Alignment of conduits for the nascent polypeptide chain in the ribosome-Sec61 complex. Science 278: 2123–2126

    Article  PubMed  CAS  Google Scholar 

  • Bernabeu C, Lake JA (1982) Nascent polypeptide chains emerge from the exit domain of the large ribosomal subunit: immune mapping of the nascent chain. Proc Natl Acad Sci USA 79: 3111–3115

    Article  PubMed  CAS  Google Scholar 

  • Bernabeu C, Tobin EM, Fowler A, Zabin I, Lake JA (1983) Nascent polypeptide chains exit the ribosome in the same relative position in both eukaryotes and prokaryotes. J Cell Biol 96: 1471–1474

    Article  PubMed  CAS  Google Scholar 

  • Berndt U, Oellerer S, Zhang Y, Johnson AE, Rospert S (2009) A signal-anchor sequence stimulates signal recognition particle binding to ribosomes from inside the exit tunnel. Proc Natl Acad Sci USA 106: 1398–1403

    Article  PubMed  Google Scholar 

  • Bhushan S, Gartmann M, Halic M, Armache JP, Jarasch A, Mielke T, Berninghausen O, Wilson DN, Beckmann R (2010) alpha-Helical nascent polypeptide chains visualized within distinct regions of the ribosomal exit tunnel. Nat Struct Mol Biol 17: 313–317

    Article  PubMed  CAS  Google Scholar 

  • Blobel G, Sabatini DD (1970) Controlled proteolysis of nascent polypeptides in rat liver cell fractions. I. Location of the polypeptides within ribosomes. J Cell Biol 45: 130–145

    Article  PubMed  CAS  Google Scholar 

  • Cabrita LD, Dobson CM, Christodoulou J (2010) Protein folding on the ribosome. Curr Opin Struct Biol 20: 33–45

    Article  PubMed  CAS  Google Scholar 

  • Chandramouli P, Topf M, Menetret JF, Eswar N, Cannone JJ, Gutell RR, Sali A, Akey CW (2008) Structure of the mammalian 80S ribosome at 8.7 A resolution. Structure 16: 535–548

    Article  PubMed  CAS  Google Scholar 

  • Choi K, Brimacombe R (1998) The path of the growing peptide chain through the 23S rRNA in the 50S ribosomal subunit; a comparative cross-linking study with three different peptide families. Nucleic Acids Res 26: 887–895

    Article  PubMed  CAS  Google Scholar 

  • Cruz-Vera L, Rajagopal S, Squires C, Yanofsky C (2005) Features of ribosome-peptidyl-tRNA interactions essential for tryptophan induction of tna operon expression. Mol Cell 19: 333–343

    Article  PubMed  CAS  Google Scholar 

  • Cruz-Vera LR, Gong M, Yanofsky C (2006) Changes produced by bound tryptophan in the ribosome peptidyl transferase center in response to TnaC, a nascent leader peptide. Proc Natl Acad Sci USA 103: 3598–3603

    Article  PubMed  CAS  Google Scholar 

  • Cruz-Vera LR, New A, Squires C, Yanofsky C (2007) Ribosomal features essential for tna operon induction: tryptophan binding at the peptidyl transferase center. J Bacteriol 189: 3140–3146

    Article  PubMed  CAS  Google Scholar 

  • Cruz-Vera LR, Yanofsky C (2008) Conserved residues Asp16 and Pro24 of TnaC-tRNAPro participate in tryptophan induction of Tna operon expression. J Bacteriol 190: 4791–4797

    Article  PubMed  CAS  Google Scholar 

  • de Bakker PI, Furnham N, Blundell TL, DePristo MA (2006) Conformer generation under restraints. Curr Opin Struct Biol 16: 160–165

    Article  CAS  Google Scholar 

  • Deutsch C (2003) The birth of a channel. Neuron 40: 265–276

    Article  PubMed  CAS  Google Scholar 

  • Evans MS, Sander IM, Clark PL (2008) Cotranslational folding promotes beta-helix formation and avoids aggregation in vivo. J Mol Biol 383: 683–692

    Article  PubMed  CAS  Google Scholar 

  • Frank J, Zhu J, Penczek P, Li YH, Srivastava S, Verschoor A, Radermacher M, Grassucci R, Lata RK, Agrawal RK (1995) A model of protein synthesis based on cryo-electron microscopy of the E. coli ribosome. Nature 376: 441–444

    Article  PubMed  CAS  Google Scholar 

  • Gong F, Ito K, Nakamura Y, Yanofsky C (2001) The mechanism of tryptophan induction of tryptophanase operon expression: tryptophan inhibits release factor-mediated cleavage of TnaC-peptidyl-tRNA(Pro). Proc Natl Acad Sci USA 98: 8997–9001

    Article  PubMed  CAS  Google Scholar 

  • Gong F, Yanofsky C (2002) Instruction of translating ribosome by nascent peptide. Science 297: 1864–1867

    Article  PubMed  CAS  Google Scholar 

  • Halic M, Blau M, Becker T, Mielke T, Pool MR, Wild K, Sinning I, Beckmann R (2006 a) Following the signal sequence from ribosomal tunnel exit to signal recognition particle. Nature 444: 507–511

    Article  PubMed  CAS  Google Scholar 

  • Halic M, Gartmann M, Schlenker O, Mielke T, Pool MR, Sinning I, Beckmann R (2006b) Signal recognition particle receptor exposes the ribosomal translocon binding site. Science 312: 745–747

    Article  PubMed  CAS  Google Scholar 

  • Harms J, Schluenzen F, Zarivach R, Bashan A, Gat S, Agmon I, Bartels H, Franceschi F, Yonath A (2001) High resolution structure of the large ribosomal subunit from a mesophilic eubacterium. Cell 107: 679–688

    Article  PubMed  CAS  Google Scholar 

  • Jewett MC, Swartz JR (2004) Mimicking the Escherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis. Biotechnol Bioeng 86: 19–26

    Article  PubMed  CAS  Google Scholar 

  • Jin H, Kelley AC, Loakes D, Ramakrishnan V (2010) Structure of the 70S ribosome bound to release factor 2 and a substrate analog provides insights into catalysis of peptide release. Proc Natl Acad Sci USA 107: 8593–8598

    Article  PubMed  Google Scholar 

  • Korostelev A, Asahara H, Lancaster L, Laurberg M, Hirschi A, Zhu J, Trakhanov S, Scott WG, Noller HF (2008) Crystal structure of a translation termination complex formed with release factor RF2. Proc Natl Acad Sci USA 105: 19 684–19 689

    Article  Google Scholar 

  • Kosolapov A, Deutsch C (2009) Tertiary interactions within the ribosomal exit tunnel. Nat Struct Mol Biol 16: 405–411

    Article  PubMed  CAS  Google Scholar 

  • Kosolapov A, Tu L, Wang J, Deutsch C (2004) Structure acquisition of the T1 domain of Kv1.3 during biogenesis. Neuron 44: 295–307

    Article  PubMed  CAS  Google Scholar 

  • Kramer G, Boehringer D, Ban N, Bukau B (2009) The ribosome as a platform for co-translational processing, folding and targeting of newly synthesized proteins. Nat Struct Mol Biol 16: 589–597

    Article  PubMed  CAS  Google Scholar 

  • Kramer G, Ramachandiran V, Hardesty B (2001) Cotranslational folding—omnia mea mecum porto? Int J Biochem Cell Biol 33: 541–553

    Article  PubMed  CAS  Google Scholar 

  • Laurberg M, Asahara H, Korostelev A, Zhu J, Trakhanov S, Noller HF (2008) Structural basis for translation termination on the 70S ribosome. Nature 454: 852–857

    Article  PubMed  CAS  Google Scholar 

  • Liao SR, Lin JL, Do H, Johnson AE (1997) Both lumenal and cytosolic gating of the aqueous ER translocon pore are regulated from inside the ribosome during membrane protein integration. Cell 90: 31–41

    Article  PubMed  CAS  Google Scholar 

  • Lim VI, Spirin AS (1986) Stereochemical analysis of ribosomal transpeptidation. Conformation of nascent peptide. J Mol Biol 188: 565–574

    Article  PubMed  CAS  Google Scholar 

  • Liu DV, Zawada JF, Swartz JR (2005) Streamlining Escherichia coli S30 extract preparation for economical cell-free protein synthesis. Biotechnol Prog 21: 460–465

    Article  PubMed  CAS  Google Scholar 

  • Lovett PS, Rogers EJ (1996) Ribosome regulation by the nascent peptide. Microbiol Rev 60: 366–385

    PubMed  CAS  Google Scholar 

  • Lu J, Deutsch C (2005a) Folding zones inside the ribosomal exit tunnel. Nat Struct Mol Biol 12: 1123–1129

    Article  PubMed  CAS  Google Scholar 

  • Lu J, Deutsch C (2005b) Secondary structure formation of a transmembrane segment in Kv channels. Biochemistry 44: 8230–8243

    Article  PubMed  CAS  Google Scholar 

  • Lu J, Deutsch C (2008) Electrostatics in the ribosomal tunnel modulate chain elongation rates. J Mol Biol 384: 73–86

    Article  PubMed  CAS  Google Scholar 

  • Lu J, Kobertz WR, Deutsch C (2007) Mapping the electrostatic potential within the ribosomal exit tunnel. J Mol Biol 371: 1378–1391

    Article  PubMed  CAS  Google Scholar 

  • Malkin LI, Rich A (1967) Partial resistance of nascent polypeptide chains to proteolytic digestion due to ribosomal shielding. J Mol Biol 26: 329–346

    Article  PubMed  CAS  Google Scholar 

  • Marqusee S, Baldwin RL (1987) Helix stabilization by Glu-... Lys+ salt bridges in short peptides of de novo design. Proc Natl Acad Sci USA 84: 8898–8902

    Article  PubMed  CAS  Google Scholar 

  • Milligan RA, Unwin PNT (1986) Location of exit channel for nascent protein in 80S ribosome. Nature 319: 693–695

    Article  PubMed  CAS  Google Scholar 

  • Mingarro I, Nilsson I, Whitley P, von Heijne G (2000) Different conformations of nascent polypeptides during translocation across the ER membrane. BMC Cell Biol 1: 3

    Article  PubMed  CAS  Google Scholar 

  • Morgan DG, Menetret JF, Radermacher M, Neuhof A, Akey IV, Rapoport TA, Akey CW (2000) A comparison of the yeast and rabbit 80 S ribosome reveals the topology of the nascent chain exit tunnel, inter-subunit bridges and mammalian rRNA expansion segments. J Mol Biol 301: 301–321

    Article  PubMed  CAS  Google Scholar 

  • Morris DR, Geballe AP (2000) Upstream open reading frames as regulators of mRNA translation. Mol Cell Biol 20: 8635–8642

    Article  PubMed  CAS  Google Scholar 

  • Nakatogawa H, Ito K (2002) The ribosomal exit tunnel functions as a discriminating gate. Cell 108: 629–636

    Article  PubMed  CAS  Google Scholar 

  • Nissen P, Hansen J, Ban N, Moore PB, Steitz TA (2000) The structural basis of ribosome activity in peptide bond synthesis. Science 289: 920–930

    Article  PubMed  CAS  Google Scholar 

  • Picking WD, Picking WL, Odom OW, Hardesty B (1992) Fluorescence characterization of the environment encountered by nascent polyalanine and polyserine as they exit Escherichia coli ribosomes during translation. Biochemistry 31: 2368–2375

    Article  PubMed  CAS  Google Scholar 

  • Polacek N, Gomez MJ, Ito K, Xiong L, Nakamura Y, Mankin A (2003) The critical role of the universally conserved A2602 of 23S ribosomal RNA in the release of the nascent peptide during translation termination. Mol Cell 11: 103–112

    Article  PubMed  CAS  Google Scholar 

  • Polacek N, Mankin AS (2005) The ribosomal peptidyl transferase center: structure, function, evolution, inhibition. Crit Rev Biochem Mol Biol 40: 285–311

    Article  PubMed  CAS  Google Scholar 

  • Ramachandiran V, Willms C, Kramer G, Hardesty B (2000) Fluorophores at the N terminus of nascent chloramphenicol acetyltransferase peptides affect translation and movement through the ribosome. J Biol Chem 275: 1781–1786

    Article  PubMed  CAS  Google Scholar 

  • Schmeing TM, Huang KS, Kitchen DE, Strobel SA, Steitz TA (2005a) Structural insights into the roles of water and the 2′ hydroxyl of the P site tRNA in the peptidyl transferase reaction. Mol Cell 20: 437–448

    Article  PubMed  CAS  Google Scholar 

  • Schmeing TM, Huang KS, Strobel SA, Steitz TA (2005b) An induced-fit mechanism to promote peptide bond formation and exclude hydrolysis of peptidyl-tRNA. Nature 438: 520–524

    Article  PubMed  CAS  Google Scholar 

  • Schuwirth B, Borovinskaya M, Hau C, Zhang W, Vila-Sanjurjo A, Holton J, Cate J (2005) Structures of the bacterial ribosome at 3.5 Å resolution. Science 310: 827–834

    Article  PubMed  CAS  Google Scholar 

  • Seidelt B, Innis CA, Wilson DN, Gartmann M, Armache JP, Villa E, Trabuco LG, Becker T, Mielke T, Schulten K, Steitz TA, Beckmann R (2009) Structural insight into nascent polypeptide chain-mediated translational stalling. Science 326: 1412–1415

    Article  PubMed  CAS  Google Scholar 

  • Selmer M, Dunham C, Murphy Ft, Weixlbaumer A, Petry S, Kelley A, Weir J, Ramakrishnan V (2006) Structure of the 70S ribosome complexed with mRNA and tRNA. Science 313: 1935–1942

    Article  PubMed  CAS  Google Scholar 

  • Simonovic M, Steitz TA (2009) A structural view on the mechanism of the ribosome-catalyzed peptide bond formation. Biochim Biophys Acta 1789: 612–623

    Article  PubMed  CAS  Google Scholar 

  • Smith WP, Tai PC, Davis BD (1978) Interaction of secreted nascent chains with surrounding membrane in Bacillus subtilis. Proc Natl Acad Sci USA 75: 5922–5925

    Article  PubMed  CAS  Google Scholar 

  • Taylor DJ, Devkota B, Huang AD, Topf M, Narayanan E, Sali A, Harvey SC, Frank J (2009) Comprehensive molecular structure of the eukaryotic ribosome. Structure 17: 1591–1604

    Article  PubMed  CAS  Google Scholar 

  • Tenson T, Ehrenberg M (2002) Regulatory nascent peptides in the ribosomal tunnel. Cell 108: 591–594

    Article  PubMed  CAS  Google Scholar 

  • Trabuco LG, Villa E, Mitra K, Frank J, Schulten K (2008) Flexible fitting of atomic structures into electron microscopy maps using molecular dynamics. Structure 16: 673–683

    Article  PubMed  CAS  Google Scholar 

  • Tsalkova T, Odom OW, Kramer G, Hardesty B (1998) Different conformations of nascent peptides on ribosomes. J Mol Biol 278: 713–723

    Article  PubMed  CAS  Google Scholar 

  • Tu LW, Deutsch C (2010) A folding zone in the ribosomal exit tunnel for Kv1.3 helix formation. J Mol Biol 396: 1346–1360

    Article  PubMed  CAS  Google Scholar 

  • Vazquez-Laslop N, Thum C, Mankin AS (2008) Molecular mechanism of drug-dependent ribosome stalling. Mol Cell 30: 190–202

    Article  PubMed  CAS  Google Scholar 

  • Voss NR, Gerstein M, Steitz TA, Moore PB (2006) The geometry of the ribosomal polypeptide exit tunnel. J Mol Biol 360: 893–906

    Article  PubMed  CAS  Google Scholar 

  • Weixlbaumer A, Jin H, Neubauer C, Voorhees R, Petry S, Kelley A, Ramakrishnan V (2008) Insights into translational termination from the structure of RF2 bound to the ribosome. Science 322: 953–956

    Article  PubMed  CAS  Google Scholar 

  • Wilson DN, Schluenzen F, Harms JM, Yoshida T, Ohkubo T, Albrecht R, Buerger J, Kobayashi Y, Fucini P (2005) X-ray crystallography study on ribosome recycling: the mechanism of binding and action of RRF on the 50S ribosomal subunit. EMBO J 24: 251–260

    Article  PubMed  CAS  Google Scholar 

  • Woolhead CA, McCormick PJ, Johnson AE (2004) Nascent membrane and secretory proteins differ in FRET-detected folding far inside the ribosome and in their exposure to ribosomal proteins. Cell 116: 725–736

    Article  PubMed  CAS  Google Scholar 

  • Yang R, Cruz-Vera LR, Yanofsky C (2009) 23S rRNA nucleotides in the peptidyl transferase center are essential for tryptophanase operon induction. J Bacteriol 191: 3445–3450

    Article  PubMed  CAS  Google Scholar 

  • Yap MN, Bernstein HD (2009) The plasticity of a translation arrest motif yields insights into nascent polypeptide recognition inside the ribosome tunnel. Mol Cell 34: 201–211

    Article  PubMed  CAS  Google Scholar 

  • Yonath A, Leonard KR, Wittmann HG (1987) A tunnel in the large ribosomal subunit revealed by three-dimensional image reconstruction. Science 236: 813–816

    Article  PubMed  CAS  Google Scholar 

  • Youngman EM, Brunelle JL, Kochaniak AB, Green R (2004) The active site of the ribosome is composed of two layers of conserved nucleotides with distinct roles in peptide bond formation and peptide release. Cell 117: 589–599

    Article  PubMed  CAS  Google Scholar 

  • Yusupov MM, Yusupova GZ, Baucom A, Lieberman K, Earnest TN, Cate JH, Noller HF (2001) Crystal structure of the ribosome at5.5 A resolution. Science 292: 883–896

    Article  PubMed  CAS  Google Scholar 

Download references

Authors
  1. Daniel N. Wilson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shashi Bhushan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Thomas Becker
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Roland Beckmann
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, 37077, Göttingen, Germany

    Marina V. Rodnina  & Wolfgang Wintermeyer  & 

  2. Howard Hughes Medical Institute Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA

    Rachel Green

Rights and permissions

Reprints and permissions

Copyright information

© 2011 Springer-Verlag/Wien

About this chapter

Cite this chapter

Wilson, D.N., Bhushan, S., Becker, T., Beckmann, R. (2011). Nascent polypeptide chains within the ribosomal tunnel analyzed by cryo-EM. In: Rodnina, M.V., Wintermeyer, W., Green, R. (eds) Ribosomes. Springer, Vienna. https://doi.org/10.1007/978-3-7091-0215-2_31

Download citation

Publish with us

Policies and ethics

Search

Navigation