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Chapter 24 : Folding of Nascent Peptides on Ribosomes

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Abstract:

This chapter reviews nascent peptides on ribosomes and how the ribosome may contribute to the folding of nascent proteins. An open space, a tunnel, inside the 50S ribosomal subunit was first detected in the early analyses of crystalline arrays of ribosomes. The recognition of a tunnel through the 50S subunit led to the suggestion that this might be the path followed by the nascent peptide through the ribosome. However, recent results strongly favor the conclusion that the path of the nascent peptide is a tunnel rather than a channel on the surface of the large subunit. Noller and coworkers reported peptide bond formation by the RNA portion of the 50S ribosomal subunit. Proteins are synthesized vectorially from their N termini to their C termini on ribosomes. Nascent globin appears to constitute an exception to the principle that nascent proteins cannot fold into the native conformation. An accumulation of a heterogeneous band of relatively small peptides occurs during the synthesis of chloramphenicol acetyltransferase (CAT) with coumarin-labeled initiator tRNA. Rhodanese enzymatic activity was determined after coupled transcription-translation in the absence of the factor and in its presence and in the presence of the activating fraction after it had been preincubated for 10 min at elevated temperatures.

Citation: Hardesty B, Kramer G, Tsalkova T, Ramachandiran V, McIntosh B, Brod D. 2000. Folding of Nascent Peptides on Ribosomes, p 287-298. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch24

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Figure 1

Translational pause sites differ for different proteins. Six different proteins were synthesized by coupled transcription-translation in the cell-free system with a small amount of the A19 S30 fraction (cf. ). The coding sequences were in plasmids under the control of the T7 promoter. [C]leucine was the radioactive precursor. After coupled transcription- translation, an aliquot (15 µl) was withdrawn to determine the amount of polypeptides formed, another aliquot (15 µl) of the reaction mixtures was analyzed by polyacrylamide gel electrophoresis according to the method of , and the radioactive bands were visualized by phosphorimaging. Lane 1, CAT ( , ˜25,600; 13 leucines; 207 pmol of leucine incorporated); lane 2, hamster rhodanese ( , ˜33,000; 28 leucines; 103 pmol of leucine incorporated); lane 3, bovine rhodanese ( , ˜33,000; 25 leucines; 115 pmol of leucine incorporated); lane 4, trigger factor ( , ˜58,000; 31 leucines; 73 pmol of leucine incorporated); lane 5, release factor 1 ( , ˜40,500; 32 leucines; 50 pmol of leucine incorporated); lane 6, release factor 2 ( , ˜41,200; 29 leucines; 61 pmol of leucine incorporated).

Citation: Hardesty B, Kramer G, Tsalkova T, Ramachandiran V, McIntosh B, Brod D. 2000. Folding of Nascent Peptides on Ribosomes, p 287-298. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch24
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Figure 2

Puromycin reactivity of nascent peptides. Hamster (H) and bovine (B) rhodanese were synthesized as described in the legend to Fig. 1 except that 5 µl of S30 ( MRE 600) and nonradioactive amino acids were used. After coupled transcription-translation, P-labeled C-puro was added to give 8 µM, and the incubation continued for 10 min. Then one aliquot was used to determine the incorporation of C-puro into nascent peptides, and another aliquot was processed for polyacrylamide gel electrophoresis and phosphorimaging. The result is shown. Lane 1, no plasmid added; lane 2, hamster rhodanese; lane 3, bovine rhodanese. About 4.5 and 5 pmol of C-puro was incorporated into hamster and bovine polypeptides, respectively. The numbers on the left side of the gel indicate the numbers of amino acids (a a #) corresponding to the puromycin-labeled band in either lane 2 (H) or lane 3 (B). On the right side of the gel, amino acid positions in which a change in amino acid composition from H to B occurred are given.

Citation: Hardesty B, Kramer G, Tsalkova T, Ramachandiran V, McIntosh B, Brod D. 2000. Folding of Nascent Peptides on Ribosomes, p 287-298. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch24
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Figure 4

Structures of different fluorophore–Met-tRNA species that were synthesized and used in coupled transcription-translation.

Citation: Hardesty B, Kramer G, Tsalkova T, Ramachandiran V, McIntosh B, Brod D. 2000. Folding of Nascent Peptides on Ribosomes, p 287-298. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch24
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Image of Figure 5
Figure 5

Time course of CAT synthesis with fluorophore–Met-tRNA. CAT was synthesized by coupled transcription-translation with a reduced amount of the S30 fraction (2.5 µl / 30-µl assay). Protein synthesis was initiated with either f[S]Met-tRNA, cascade yellow–[S]Met-tRNA, pyrene–[S]Met-tRNA, coumarin–[S]Met-tRNA, or eosin–[S]Met-tRNA. The reaction mixtures were incubated, and at the indicated times aliquots were withdrawn and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and phosphorimaging. The results are shown.

Citation: Hardesty B, Kramer G, Tsalkova T, Ramachandiran V, McIntosh B, Brod D. 2000. Folding of Nascent Peptides on Ribosomes, p 287-298. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch24
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Figure 3

Fusidic acid keeps short CAT peptides in the ribosomal P site. CAT was synthesized for either 5 min (lanes 1 and 2) or 30 min (lanes 3 and 4) by coupled transcription-translation with an S30 fraction from MRE 600 and unlabeled amino acids. Then, either HO (lanes 1 and 3) or fusidic acid (lanes 2 and 4) was added and the samples were kept on ice for 3 min before the addition of P-labeled C-puro. The samples were incubated and processed as described in the legend to Fig. 2. The numbers on the left refer to positions of molecular weight markers as follows: 31, carbonic anhydrase; 20, soybean trypsin inhibitor; 14, lysozyme; 6, aprotinin; 3.5, insulin chain. +, present; −, absent.

Citation: Hardesty B, Kramer G, Tsalkova T, Ramachandiran V, McIntosh B, Brod D. 2000. Folding of Nascent Peptides on Ribosomes, p 287-298. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch24
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Image of Figure 6
Figure 6

Interaction of eosin–Met-tRNA with IF2 and its binding to salt-washed ribosomes. (A) Binding of f[S]Met-tRNA and eosin–[S]Met-tRNA to IF2 was determined by the Millipore filter binding assay in the absence of Mg ( ). IF2 was isolated according to the method of from an strain transformed by a plasmid containing the IF2 sequence. (We thank U. RajBhandari for providing the plasmid.) (B) Binding of f[S]Met-tRNA and eosin–[S]Met-tRNA to salt-washed ribosomes was carried out in the presence of 18 mM Mg in the absence (w/o) or presence (w/ ) of an excess of IF2.

Citation: Hardesty B, Kramer G, Tsalkova T, Ramachandiran V, McIntosh B, Brod D. 2000. Folding of Nascent Peptides on Ribosomes, p 287-298. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch24
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Figure 7

Increased synthesis of full-length protein in the S30 fraction from MRE 600. Both RHO and CAT were synthesized for 20 min in the presence of [C]Leu. The samples shown in lanes 2 to 4 received increasing amounts of a fraction derived from a different S30 before protein synthesis was started. This fraction was isolated by S300 chromatography and eluted well behind ribosomes but before ˜100-kDa proteins. The arrows indicate the positions of the full-length proteins.

Citation: Hardesty B, Kramer G, Tsalkova T, Ramachandiran V, McIntosh B, Brod D. 2000. Folding of Nascent Peptides on Ribosomes, p 287-298. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch24
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References

/content/book/10.1128/9781555818142.chap24
1. Agrawal, R. K.,, P. Penczek,, R. A. Grassucci,, and J. Frank. 1998. Visualization of elongation factor G on the Escherichia coli ribosome: the mechanism of translocation. Proc. Natl. Acad. Sci. USA 95:61346138.
2. Ban, N.,, B. Freeborn,, P. Nissen,, P. Penczek,, R. A. Grassucci,, R. Sweet,, J. Frank,, P. B. Moore,, and T. A. Steitz. 1998. A 9 Å resolution x-ray crystallographic map of the large ribosomal subunit. Cell 93:11051116.
3. Beckman, R.,, D. Bubeck,, R. Grassucci,, P. Penzcek,, A. Verschoor,, G. Blobel,, and J. Frank. 1997. Alignments of conduits for the nascent polypeptide chain in the ribosome-Sec61 complex. Science 278:21232126.
4. Bernabeu, C.,, and J. A. Lake. 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 79:31113115.
5. Chantrenne, H., 1961. The biosynthesis of proteins, p. 122147. In P. Alexander, and Z. Bacq (ed.), Modern Trends in Physiological Science, vol. 14. Pergamon Press, Elmsford, N.Y.
6. Chattopadhyay, S.,, S. Pal,, D. Pal,, D. Sarkar,, C. Suparna,, and C. Das Gupta. 1999. Protein folding in Escherichia coli: role of 23S ribosomal RNA. Biochim. Biophys. Acta 1429:293298.
7. Choi, K. M.,, and R. Brimacombe. 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. 26:887895.
8. Choi, K. M.,, J. F. Atkins,, R. F. Gesteland,, and R. Brimacombe. 1998. Flexibility of the nascent polypeptide chain within the ribosome. Contacts from the peptide N-terminus to the 30S subunit. Eur. J. Biochem. 255:409413.
9. Das, B.,, S. Chattopadhyay,, A. K. Bera,, and C. Dasgupta. 1996. In vitro protein folding by ribosomes from Escherichia coli, wheat germ and rat liver. The role of the 50S particle and its 23S rRNA. Eur. J. Biochem. 235:613621.
10. Eisenstein, M.,, B. Hardesty,, O. W. Odom,, W. Kudlicki,, G. Kramer,, T. Arad,, F. Franceschi,, and A. Yonath,. 1994. Modeling and experimental study of the progression of nascent proteins in ribosomes, p. 213246. In G. Pifat (ed.), Supramolecular Structure and Function. Ruder Boskovic Institute, Zagreb, Croatia.
11. Ellis, R. J. 1997. Do molecular chaperones have to be proteins? Biochem. Biophys. Res. Commun. 238:687692.
12. Ewalt, K. L.,, J. P. Hendrick,, W. A. Houry,, and F. U. Hartl. 1997. In vivo observation of polypeptide flux through the bacterial chaperonin system. Cell 90:491500.
13. Frank, J. 1998. The ribosome—structure and functional ligand-binding experiments using cryo-electron microscopy. J. Struct. Biol. 124:142150.
14. Frank, J.,, J. Zhu,, P. Penczek,, Y. Li,, S. Srivastava,, A. Verschoor,, M. Radermacher,, R. Grassucci,, R. K. Lata,, and R. K. Agrawal. 1995. A model of protein synthesis based on cryo-electron microscopy of the E. coli ribosome. Nature 376:441444.
15. Green, R.,, and H. F. Noller. 1997. Ribosomes and translation. Annu. Rev. Biochem. 66:679716.
16. Green, R.,, C. Switzer,, and H. F. Noller. 1998. Ribosome-catalyzed peptide-bond formation with an A-site substrate covalently linked to 23S RNA. Science 280:286289.
17. Hardesty, B.,, T. Tsalkova,, and G. Kramer. 1999. Co-translational folding. Curr. Opin. Struct. Biol. 9:111114.
18. Hesterkamp, T.,, S. Hauser,, H. Lütcke,, and B. Bukau. 1996. Escherichia coli trigger factor is a prolyl isomerase that associates with nascent peptide chains. Proc. Natl. Acad. Sci. USA 93:44374441.
19. Heurgué-Hamard, V.,, R. Karimi,, L. Mora,, J. MacDougall,, C. Leboeuf,, G. Grentzmann,, M. Ehrenberg,, and R. H. Buckingham. 1998. Ribosome release factor RF4 and termination factor RF3 are involved in dissociation of peptidyl-tRNA from the ribosome. EMBO J. 17:808816.
20. Komar, A. A.,, A. Kommer,, I. A. Krasheninnikov,, and A. S. Spirin. 1997. Cotranslational folding of globin. J. Biol. Chem. 272:1064610651.
21. Kudlicki, W.,, J. Chirgwin,, G. Kramer,, and B. Hardesty. 1995. Folding of an enzyme into an active conformation while bound as peptidyl-tRNA to the ribosome. Biochemistry 34:1428414287.
22. Kudlicki, W.,, O. W. Odom,, G. Kramer,, and B. Hardesty. 1996. Binding of an N-terminal rhodanese peptide to DnaJ and to ribosomes. J. Biol. Chem. 271:3116031165.
23. Kudlicki, W.,, A. Coffman,, G. Kramer,, and B. Hardesty. 1997. Ribosomes and ribosomal RNA as chaperones. Fold. Des. 2:101108.
24. Lim, V. I.,, and A. S. Spirin. 1986. Stereochemical analysis of ribosomal transpeptidation. Conformation of nascent peptide. J. Mol. Biol. 188:565577.
25. Lorimer, G. H. 1996. A quantitative assessment of the role of chaperonin proteins in protein folding in vivo. FASEB J. 10:59.
26. Makeyev, E. V.,, V. A Kolb,, and A. S. Spirin. 1996. Enzymatic activity of the ribosome-bound nascent polypeptide. FEBS Lett. 376:166170.
27. Malhotra, A.,, P. Penczek,, R. Agrawal,, I. S. Gabashvili,, R. A. Grassucci,, R. A. Junemann,, N. Burkhardt,, K. Nierhaus,, and J. Frank. 1998. Escherichia coli 70 S ribosome at 15Å resolution by cryo-electron microscopy: localization of fMet-tRNAfMet and fitting of L1 protein. J. Mol. Biol. 280:103116.
28. Miller, D. M.,, R. Delgado,, J. M. Chirgwin,, S. C. Hardies,, and P. M. Horowitz. 1991. Expression of cloned bovine adrenal rhodanese. J. Biol. Chem. 266:46864691.
29. Miller, S. P.,, and J. W. Bodley. 1991. α-Sarcin cleavage of ribosomal RNA is inhibited by the binding of elongation factor G or thiostrepton to the ribosome. Nucleic Acid Res. 19:16571660.
30. Moazed, D.,, and H. F. Noller. 1987. Chloramphenicol, erythromycin, carbomycin and vernamycin B protect overlapping sites in the peptidyl transferase region of 23S ribosomal RNA. Biochimie 69:879884.
31. Mortensen, K. K.,, N. R. Nyengaard,, J. W. B. Hershey,, S. Laalami,, and H. U. Sperling-Petersen. 1991. Superexpression and fast purification of E. coli initiation factor IF-2. Biochimie 73:983989.
32. Nierhaus K. H., , S. Schilling-Bartetzko, , and T. Twardowski. 1992. The two main states of the elongating ribosome and the role of the α-sarcin stem loop structure of 23S RNA. Biochimie 74:403410.
33. Picking, W. D.,, O. W. Odom,, T. Tsalkova,, I. Serdyuk,, and B. Hardesty. 1991. The conformation of nascent polylysine and polyphenylalanine peptides on ribosomes. J. Biol. Chem. 266:15341542.
34. Picking, W. D.,, O. W. Odom,, and B. Hardesty. 1992a. Evidence for RNA in the peptidyl transferase center of Escherichia coli ribosomes as indicated by fluorescence. Biochemistry 31:1256512570.
35. Picking, W. D.,, W. L. Picking,, O. W. Odom,, and B. Hardesty. 1992b. Fluorescence characterization of the environment encountered by nascent polyalanine and polyserine as they exit Escherichia coli ribosomes during translation. Biochemistry 31:23682375.
36. Ramachandiran, V.,, C. Willms,, G. Kramer,, and B. Hardesty. Fluorophores at the N terminus of nascent chloramphenicol acetyltransferase peptides affect translation and movement through the ribosome. J. Biol. Chem., in press.
37. Ryabova, L. A.,, O. M. Selivanova,, V. I. Baranov,, V. D. Vasiliev,, and A. S. Spirin. 1988. Does the channel for nascent peptide exist inside the ribosome? FEBS Lett. 236:255260.
38. Schägger, M.,, and G. von Jagow. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166:368379.
39. Stoller, G.,, K. P. Rücknagel,, K. Nierhaus,, F. X. Schmid,, G. Fischer,, and J. U. Rahfeld. 1995. Identification of the peptidyl-prolyl cis/ trans isomerase bound to the Escherichia coli ribosome as the trigger factor. EMBO J. 14:49394948.
40. Sundari, R.,, E. A. Stringer,, L. H. Schulmann,, and U. Maitra. 1976. Interaction of bacterial initiation factor 2 with initiator tRNA. J. Biol. Chem. 251:33383345.
41. Thulasiraman, V.,, C. Yang,, and J. Frydman. 1999. In vivo newly translated polypeptides are sequestered in a protected folding environment. EMBO J. 18:8595.
42. Trevino, R. J.,, J. Hunt,, P. M. Horowitz,, and J. M. Chirgwin. 1995. Chinese hamster rhodanese cDNA: activity of the expressed protein is not blocked by a C-terminal extension. Protein Expr. Purif. 6:693699.
43. Tsalkova, T.,, O. W. Odom,, G. Kramer,, and B. Hardesty. 1998. Different conformations of nascent peptides on ribosomes. J. Mol. Biol. 278:713723.
44. Tsalkova, T.,, G. Kramer,, and B. Hardesty. 1999. The effect of a hydrophobic N-terminal probe on translational pausing of chloramphenicol acetyl transferase and rhodanese. J. Mol. Biol. 286: 7181.
45. Xu, Z.,, and P. B. Sigler. 1998. GroEL/GroES: structure and function of a two-stroke folding machine. J. Struct. Biol. 124:129141.
46. Yonath, A.,, and Z. Berkovitch-Yellin. 1993. Hollows, voids, gaps and tunnels in the ribosome. Curr. Opin. Struct. Biol. 3:175181.
47. Yonath, A.,, K. R. Leonard,, and H. G. Wittmann. 1987. A tunnel in the large ribosomal subunit revealed by three-dimensional image reconstruction. Science 236:813816.

Tables

Generic image for table
Table 1

Incorporation of -acyl-methionine into RHO and CAT

Citation: Hardesty B, Kramer G, Tsalkova T, Ramachandiran V, McIntosh B, Brod D. 2000. Folding of Nascent Peptides on Ribosomes, p 287-298. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch24

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