Protein Synthesis

Introduction

In all living cells translation is coupled with a range of downstream cellular processes such as protein folding, processing, targeting and translocation. The major players in these events are;

i) ribosome associated chaperones

ii) nascent chain processing enzymes

iii) the signal recognition particle (a complex that recognizes ribosomes translating membrane and secretory proteins and targets them to the membrane)

and

iv) translocon - a large multi-subunit membrane-bound complex responsible for protein translocation into or across the membranes.

With the aim to better understand the molecular machinery involved in co-translational protein folding we have determined the structure of the ribosome-associating chaperone trigger factor in complex with the large ribosomal subunit. Trigger factor adopts a unique conformation resembling a crouching dragon with separated domains forming the N-terminal ribosome-binding “tail”, the peptidyl-prolyl isomerase “head” and the C-terminal “arms”. We show that from its attachment point on the ribosome the chaperone forms a cradle below the nascent peptide exit tunnel to interact with newly synthesized proteins. This study, conducted in collaboration with the Bukau lab in Heidelberg, sheds new light on our understanding of chaperone assisted co-translational protein folding (Ferbitz et al. 2004)

As part of our studies aimed at co-translational protein targeting and translocation we determined a cryo-EM reconstruction of the complex between the /E. coli/ SRP and the translating ribosome. This reconstruction provides the first structural insight into the functioning of the bacterial SRP system. The reconstruction was interpreted using high-resolution structures of the ribosome and of the bacterial SRP providing a comprehensive structural model of the complex. The structure reveals which regions of the SRP and the ribosome are involved in the formation of the complex, and provide insight into the conformation of the SRP on the ribosome and into the conformational changes of the SRP that accompany signal sequence recognition (Schaffitzel et al 2006). In collaboration, we have also been able to obtain the cryo-EM reconstruction of the E. coli translocon bound to a ribosome nascent chain complex.

Recently we have initiated our studies of co-translational nascent chain processing by studying peptide deformylase (PDF). Synthesis of all bacterial proteins is initiated with formyl-methionine. Subsequent removal of the formyl group from the N-terminal methionine of all nascent chains is catalyzed by PDF, an essential bacterial enzyme. Although it had been known for almost four decades that this enzyme efficiently processes nascent chains, the mechanism of its co-translational action was not understood.

Our experiments provided the first evidence that PDF binds to the ribosome to facilitate co-translational processing of nascent chains. We demonstrated by biochemical and biophysical experiments that the C-terminal region of PDF is responsible for this interaction and revealed the details of the interaction by solving a crystal structure of a complex between the ribosome binding domain of PDF and the E. coli 70S ribosome (Bingel-Erlenmeyer et al. 2008). The binding of the enzyme to the large ribosomal subunit in the vicinity of the ribosomal tunnel orients the active site of PDF towards the tunnel exit where nascent polypeptides emerge.

These results, together with the structural information regarding the interaction between the trigger factor and the ribosome, obtained earlier in our laboratory, define the structural and enzymatic environment of the nascent chain as it emerges from the ribosomal exit tunnel. The structural arrangement of PDF and trigger factor also suggests that the latter serves not only as a chaperone but that its unique structural features act as a passive router that channels nascent chains to various processing enzymes.

Research Articles

• Kohler R, Boehringer D, Greber B, Bingel-Erlenmeyer R, Collinson I, Schaffitzel C, Ban N. (2009)
  YidC and Oxa1 form dimeric insertion pores on the translating ribosome.
  Mol Cell 34(3):344-53 Pubmed
• Bingel-Erlenmeyer R, Kohler R, Kramer G, Sandikci A, Antolić S, Maier T, Schaffitzel C, Wiedmann B, Bukau B, Ban N. (2008)
  A peptide deformylase-ribosome complex reveals mechanism of nascent chain processing.
  Nature 452(7183): 108-11 Pubmed
• Schaffitzel C, Oswald M, Berger I, Ishikawa T, Abrahams JP, Koerten HK, Koning RI and Ban N. (2006)
  Structure of the E. coli signal recognition particle bound to a translating ribosome.
  Nature 444(7118): 503-6 Pubmed
• Thore S, Leibundgut M, Ban N (2006)
  Structure of the eukaryotic thiamine pyrophosphate riboswitch with its regulatory ligand.
  Science. 312(5777): 1208-11. Pubmed
• Mitra K*, Schaffitzel C*, Shaikh T, Tama F, Jenni S, Brooks CL, Ban N & Frank J (2005)
  Structure of the E. coli protein-conducting channel bound to a translating ribosome.
  Nature 438(7066): 318-24. Pubmed Comment
• Maier T, Ferbitz L, Deuerling E, Ban N. (2005)
  A cradle for new proteins: trigger factor at the ribosome.
  Curr Opin Struct Biol. 15(2): 204-12. Pubmed
• Ferbitz L, Maier T, Patzelt H, Bukau B, Deuerling E and Ban N. (2004)
  Trigger Factor in Complex with the Ribosome forms a Molecular Cradle for Nascent Proteins.
  Nature 431(7008): 590-6 Pubmed Comment
• Gutmann S, Haebel P, Metzinger L, Sutter M, Felden B and Ban N. (2003)
  Crystal Structure of the tRNA domain of transfer messenger RNA in complex with SmpB.
  Nature, 424(6949): 699-703. Pubmed
• Kramer,
  G., Rauch, T., Rist, W., Vorderwulbecke, S., Patzelt, H.,
  Schulze-Specking, A., Ban, N., Deuerling, E., Bukau, B. (2002).
  L23 protein functions as a chaperone docking site on the ribosome.
  Nature 419(6903): 171-4 Pubmed Comment
• Ban, N., Nissen, P., Hansen, J., Moore, P. B., Steitz, T. A. (2000)
  The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution.
  Science 289(5481): 905-920. Pubmed Comment
• Nissen, P., Hansen, J., Ban, N., Moore, P. B., Steitz, T. A. (2000)
  The structural basis of ribosome activity in peptide bond synthesis.
  Science 289(5481): 920-930 Pubmed Comment
• Ban N, Nissen P, Hansen J, Capel M, Moore PB, Steitz TA (1999)
  Placement of protein and RNA structures into a 5 Å - resolution map of the 50S ribosomal subunit.
  Nature, 400: 841-847. Pubmed
• Ban N, Freeborn B, Nissen P, Penczek P, Grassucci RA, Sweet R, Frank J, Moore PB, Steitz TA (1998)
  A 9 Å resolution X-ray crystallographic map of the large ribosomal subunit.
  Cell, 93(7): 1105-1115. Pubmed

Reviews and Comments

• Selmer M, Liljas A. (2008)
  Exit biology: battle for the nascent chain.
  Structure 16(4):498-500. Pubmed
  
• Nudler E. (2006)
  Flipping riboswitches.
  Cell. 126(1): 19-22. Pubmed
• Sashital D.G. and Butcher S.E. (2006)
  Flipping off the riboswitch: RNA structure that controls gene expression
  ACS Chem Biol. 1(6):341-5. Pubmed
• Horwich A. (2004)
  Sight at the end of the tunnel.
  Nature, 431: 520-522. Pubmed
• Tuna R. (2004)
  Crouching dragon guards nascent proteins.
  JCB, 166 (7): 941-941. Pubmed Central
• Burroughs T. (2001)
  Building a blueprint of the cell's protein factory.
  HHMI bulletin 14(1): 13-17
• Building a Better Antibiotic.
  The New York Times. August 15, 2000. NY Times
• Pennisi E. (2000)
  Ribosome Structure Mirrors RNA World.
  Science Now. August 10, 2000. Science Now
• Scientists delve inside protein maker.
  BBC News. August 10, 2000. BBC News
• Cech T.R. (2000)
  The ribosome is a ribozyme.
  Science Magazine. 289(5481): 878-879. Pubmed
• Ein molekularer Riese im Detail.
  spektrumdirekt. August 14, 2000. spektrumdirekt
• Garrett R. (1999)
  Mechanics of the ribosome.
  Nature. 400: 811-812. Pubmed
• Pennisi E. (1999)
  Ribosome Finally Begins to Yield Its Complete Structure.
  Science. 285(5432): 1343. Pubmed