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. 1998 Mar;4(3):257–267.

Possible involvement of Escherichia coli 23S ribosomal RNA in peptide bond formation.

I Nitta 1, T Ueda 1, K Watanabe 1
PMCID: PMC1369615  PMID: 9510328

Abstract

Experimental results are presented suggesting that 23S rRNA is directly involved in the peptide bond formation usually performed on the ribosome. Although several reports have indicated that the eubacterial peptidyltransferase reaction does not necessarily require all the ribosomal proteins, the reconstitution of peptidyltransferase activity by a naked 23S rRNA without the help of any of the ribosomal proteins has not been reported previously. It is demonstrated that an E. coli 23S rRNA transcript synthesized by T7 RNA polymerase in vitro was able to promote peptide bond formation in the presence of 0.5% SDS. The reaction was inhibited by the peptidyltransferase-specific antibiotics chloramphenicol and carbomycin, and by digestion with RNases A and T1. Site-directed mutageneses at two highly conserved regions close to the peptidyltransferase center ring, G2252 to U2252 and C2507G2581 to U2507A2581, also suppressed peptide bond formation. These findings strongly suggest that 23S rRNA is the peptidyltransferase itself.

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Selected References

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  1. Agrawal R. K., Penczek P., Grassucci R. A., Li Y., Leith A., Nierhaus K. H., Frank J. Direct visualization of A-, P-, and E-site transfer RNAs in the Escherichia coli ribosome. Science. 1996 Feb 16;271(5251):1000–1002. doi: 10.1126/science.271.5251.1000. [DOI] [PubMed] [Google Scholar]
  2. Baer M. F., Arnez J. G., Guerrier-Takada C., Vioque A., Altman S. Preparation and characterization of RNase P from Escherichia coli. Methods Enzymol. 1990;181:569–582. doi: 10.1016/0076-6879(90)81152-k. [DOI] [PubMed] [Google Scholar]
  3. Bartetzko A., Nierhaus K. H. Mg2+/NH4+/polyamine system for polyuridine-dependent polyphenylalanine synthesis with near in vivo characteristics. Methods Enzymol. 1988;164:650–658. doi: 10.1016/s0076-6879(88)64075-4. [DOI] [PubMed] [Google Scholar]
  4. Cech T. R., Zaug A. J., Grabowski P. J. In vitro splicing of the ribosomal RNA precursor of Tetrahymena: involvement of a guanosine nucleotide in the excision of the intervening sequence. Cell. 1981 Dec;27(3 Pt 2):487–496. doi: 10.1016/0092-8674(81)90390-1. [DOI] [PubMed] [Google Scholar]
  5. Cerná J., Rychlík I., Jonák J. Peptidyl-transferase activity of Escherichia coli ribosomes digested by ribonuclease T 1 . Eur J Biochem. 1973 May 2;34(3):551–556. doi: 10.1111/j.1432-1033.1973.tb02794.x. [DOI] [PubMed] [Google Scholar]
  6. Davanloo P., Rosenberg A. H., Dunn J. J., Studier F. W. Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2035–2039. doi: 10.1073/pnas.81.7.2035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Franceschi F. J., Nierhaus K. H. Ribosomal proteins L15 and L16 are mere late assembly proteins of the large ribosomal subunit. Analysis of an Escherichia coli mutant lacking L15. J Biol Chem. 1990 Sep 25;265(27):16676–16682. [PubMed] [Google Scholar]
  8. Frank J., Zhu J., Penczek P., Li Y., Srivastava S., Verschoor A., Radermacher M., Grassucci R., Lata R. K., Agrawal R. K. A model of protein synthesis based on cryo-electron microscopy of the E. coli ribosome. Nature. 1995 Aug 3;376(6539):441–444. doi: 10.1038/376441a0. [DOI] [PubMed] [Google Scholar]
  9. Green R., Noller H. F. In vitro complementation analysis localizes 23S rRNA posttranscriptional modifications that are required for Escherichia coli 50S ribosomal subunit assembly and function. RNA. 1996 Oct;2(10):1011–1021. [PMC free article] [PubMed] [Google Scholar]
  10. Gregory S. T., Lieberman K. R., Dahlberg A. E. Mutations in the peptidyl transferase region of E. coli 23S rRNA affecting translational accuracy. Nucleic Acids Res. 1994 Feb 11;22(3):279–284. doi: 10.1093/nar/22.3.279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Guerrier-Takada C., Gardiner K., Marsh T., Pace N., Altman S. The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell. 1983 Dec;35(3 Pt 2):849–857. doi: 10.1016/0092-8674(83)90117-4. [DOI] [PubMed] [Google Scholar]
  12. Herschlag D., Cech T. R. Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active site. Biochemistry. 1990 Nov 6;29(44):10159–10171. doi: 10.1021/bi00496a003. [DOI] [PubMed] [Google Scholar]
  13. Hosaka H., Sakabe I., Sakamoto K., Yokoyama S., Takaku H. Sequence-specific cleavage of oligoribonucleotide capable of forming a stem and loop structure. J Biol Chem. 1994 Aug 5;269(31):20090–20094. [PubMed] [Google Scholar]
  14. Hüttenhofer A., Noller H. F. Hydroxyl radical cleavage of tRNA in the ribosomal P site. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):7851–7855. doi: 10.1073/pnas.89.17.7851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lake J. A. Ribosomal subunit orientations determined in the monomeric ribosome by single and by double-labeling immune electron microscopy. J Mol Biol. 1982 Oct 15;161(1):89–106. doi: 10.1016/0022-2836(82)90280-7. [DOI] [PubMed] [Google Scholar]
  16. Moazed D., Noller H. F. Chloramphenicol, erythromycin, carbomycin and vernamycin B protect overlapping sites in the peptidyl transferase region of 23S ribosomal RNA. Biochimie. 1987 Aug;69(8):879–884. doi: 10.1016/0300-9084(87)90215-x. [DOI] [PubMed] [Google Scholar]
  17. Moazed D., Noller H. F. Interaction of tRNA with 23S rRNA in the ribosomal A, P, and E sites. Cell. 1989 May 19;57(4):585–597. doi: 10.1016/0092-8674(89)90128-1. [DOI] [PubMed] [Google Scholar]
  18. Nierhaus K. H., Dohme F. Total reconstitution of functionally active 50S ribosomal subunits from Escherichia coli. Proc Natl Acad Sci U S A. 1974 Dec;71(12):4713–4717. doi: 10.1073/pnas.71.12.4713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Nitta I., Ueda T., Nojima T., Watanabe K. Template-dependent polypeptide synthesis in a factor- and energy-free translation system promoted by pyridine. J Biochem. 1995 Oct;118(4):841–849. doi: 10.1093/oxfordjournals.jbchem.a124989. [DOI] [PubMed] [Google Scholar]
  20. Nitta I., Ueda T., Watanabe K. Template-dependent peptide formation on ribosomes catalyzed by pyridine. J Biochem. 1994 Apr;115(4):803–807. doi: 10.1093/oxfordjournals.jbchem.a124412. [DOI] [PubMed] [Google Scholar]
  21. Nojima T., Nitta I., Ueda T., Watanabe K. Pyridine-promoted factor- and energy-free peptide synthesis systems prepared from various organisms including prokaryote, eukaryote, and mitochondria. J Biochem. 1996 Jun;119(6):1076–1079. doi: 10.1093/oxfordjournals.jbchem.a021350. [DOI] [PubMed] [Google Scholar]
  22. Noller H. F., Hoffarth V., Zimniak L. Unusual resistance of peptidyl transferase to protein extraction procedures. Science. 1992 Jun 5;256(5062):1416–1419. doi: 10.1126/science.1604315. [DOI] [PubMed] [Google Scholar]
  23. Noller H. F. Peptidyl transferase: protein, ribonucleoprotein, or RNA? J Bacteriol. 1993 Sep;175(17):5297–5300. doi: 10.1128/jb.175.17.5297-5300.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Noller H. F. Ribosomal RNA and translation. Annu Rev Biochem. 1991;60:191–227. doi: 10.1146/annurev.bi.60.070191.001203. [DOI] [PubMed] [Google Scholar]
  25. Pley H. W., Flaherty K. M., McKay D. B. Three-dimensional structure of a hammerhead ribozyme. Nature. 1994 Nov 3;372(6501):68–74. doi: 10.1038/372068a0. [DOI] [PubMed] [Google Scholar]
  26. Przykorska A. Influence of modified nucleosides on tRNA structure as probed by two plant nucleases. Biochimie. 1995;77(1-2):109–112. doi: 10.1016/0300-9084(96)88113-2. [DOI] [PubMed] [Google Scholar]
  27. Raué H. A., Klootwijk J., Musters W. Evolutionary conservation of structure and function of high molecular weight ribosomal RNA. Prog Biophys Mol Biol. 1988;51(2):77–129. doi: 10.1016/0079-6107(88)90011-9. [DOI] [PubMed] [Google Scholar]
  28. Rheinberger H. J., Geigenmüller U., Wedde M., Nierhaus K. H. Parameters for the preparation of Escherichia coli ribosomes and ribosomal subunits active in tRNA binding. Methods Enzymol. 1988;164:658–670. doi: 10.1016/s0076-6879(88)64076-6. [DOI] [PubMed] [Google Scholar]
  29. Richards E. G., Lecanidou R., Geroch M. E. The kinetics of renaturation of 5-S RNA from Escherichia coli in the presence of Mg 2+ ions. Eur J Biochem. 1973 Apr;34(2):262–267. doi: 10.1111/j.1432-1033.1973.tb02755.x. [DOI] [PubMed] [Google Scholar]
  30. Samaha R. R., Green R., Noller H. F. A base pair between tRNA and 23S rRNA in the peptidyl transferase centre of the ribosome. Nature. 1995 Sep 28;377(6547):309–314. doi: 10.1038/377309a0. [DOI] [PubMed] [Google Scholar]
  31. Schilling-Bartetzko S., Franceschi F., Sternbach H., Nierhaus K. H. Apparent association constants of tRNAs for the ribosomal A, P, and E sites. J Biol Chem. 1992 Mar 5;267(7):4693–4702. [PubMed] [Google Scholar]
  32. Schulze H., Nierhaus K. H. Minimal set of ribosomal components for reconstitution of the peptidyltransferase activity. EMBO J. 1982;1(5):609–613. doi: 10.1002/j.1460-2075.1982.tb01216.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Spahn C. M., Remme J., Schäfer M. A., Nierhaus K. H. Mutational analysis of two highly conserved UGG sequences of 23 S rRNA from Escherichia coli. J Biol Chem. 1996 Dec 20;271(51):32849–32856. doi: 10.1074/jbc.271.51.32849. [DOI] [PubMed] [Google Scholar]
  34. TRAUT R. R., MONRO R. E. THE PUROMYCIN REACTION AND ITS RELATION TO PROTEIN SYNTHESIS. J Mol Biol. 1964 Oct;10:63–72. doi: 10.1016/s0022-2836(64)80028-0. [DOI] [PubMed] [Google Scholar]
  35. Ungewickell E., Garrett R. A. Native and denatured structures within the 5'-region of 16 S ribosomal RNA from Escherichia coli. FEBS Lett. 1977 Dec 1;84(1):37–42. doi: 10.1016/0014-5793(77)81052-1. [DOI] [PubMed] [Google Scholar]
  36. Waldrop M. M. Finding RNA makes proteins gives 'RNA world' a big boost. Science. 1992 Jun 5;256(5062):1396–1397. doi: 10.1126/science.1376495. [DOI] [PubMed] [Google Scholar]
  37. Weitzmann C. J., Cunningham P. R., Ofengand J. Cloning, in vitro transcription, and biological activity of Escherichia coli 23S ribosomal RNA. Nucleic Acids Res. 1990 Jun 25;18(12):3515–3520. doi: 10.1093/nar/18.12.3515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yokoyama S., Watanabe K., Miyazawa T. Dynamic structures and functions of transfer ribonucleic acids from extreme thermophiles. Adv Biophys. 1987;23:115–147. doi: 10.1016/0065-227x(87)90006-2. [DOI] [PubMed] [Google Scholar]
  39. Zawadzki V., Gross H. J. Rapid and simple purification of T7 RNA polymerase. Nucleic Acids Res. 1991 Apr 25;19(8):1948–1948. doi: 10.1093/nar/19.8.1948. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. van Tol H., Gross H. J., Beier H. Non-enzymatic excision of pre-tRNA introns? EMBO J. 1989 Jan;8(1):293–300. doi: 10.1002/j.1460-2075.1989.tb03376.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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