Skip to main content
PMC home page
RNA logoLink to RNA
. 2001 Oct;7(10):1403–1415.

pH-dependent conformational flexibility within the ribosomal peptidyl transferase center.

G W Muth 1, L Chen 1, A B Kosek 1, S A Strobel 1
PMCID: PMC1370184  PMID: 11680845

Abstract

A universally conserved adenosine, A2451, within the ribosomal peptidyl transferase center has been proposed to act as a general acid-base catalyst during peptide bond formation. Evidence in support of this proposal came from pH-dependent dimethylsulfate (DMS) modification within Escherichia coli ribosomes. A2451 displayed reactivity consistent with an apparent acidity constant (pKa) near neutrality, though pH-dependent structural flexibility could not be rigorously excluded as an explanation for the enhanced reactivity at high pH. Here we present three independent lines of evidence in support of the alternative interpretation. First, A2451 in ribosomes from the archaebacteria Haloarcula marismortui displays an inverted pH profile that is inconsistent with proton-mediated base protection. Second, in ribosomes from the yeast Saccharomyces cerevisiae, C2452 rather than A2451 is modified in a pH-dependent manner. Third, within E. coli ribosomes, the position of A2451 modification (N1 or N3 imino group) was analyzed by testing for a Dimroth rearrangement of the N1-methylated base. The data are more consistent with DMS modification of the A2451 N1, a functional group that, according to the 50S ribosomal crystal structure, is solvent inaccessible without structural rearrangement. It therefore appears that pH-dependent DMS modification of A2451 does not provide evidence either for or against a general acid-base mechanism of protein synthesis. Instead the data suggest that there is pH-dependent conformational flexibility within the peptidyl transferase center, the exact nature and physiological relevance of which is not known.

Full Text

The Full Text of this article is available as a PDF (1.3 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ban N., Freeborn B., Nissen P., Penczek P., Grassucci R. A., Sweet R., Frank J., Moore P. B., Steitz T. A. A 9 A resolution X-ray crystallographic map of the large ribosomal subunit. Cell. 1998 Jun 26;93(7):1105–1115. doi: 10.1016/s0092-8674(00)81455-5. [DOI] [PubMed] [Google Scholar]
  2. Ban N., Nissen P., Hansen J., Moore P. B., Steitz T. A. The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. Science. 2000 Aug 11;289(5481):905–920. doi: 10.1126/science.289.5481.905. [DOI] [PubMed] [Google Scholar]
  3. Barras F., Marinus M. G. The great GATC: DNA methylation in E. coli. Trends Genet. 1989 May;5(5):139–143. doi: 10.1016/0168-9525(89)90054-1. [DOI] [PubMed] [Google Scholar]
  4. Bayfield M. A., Dahlberg A. E., Schulmeister U., Dorner S., Barta A. A conformational change in the ribosomal peptidyl transferase center upon active/inactive transition. Proc Natl Acad Sci U S A. 2001 Aug 21;98(18):10096–10101. doi: 10.1073/pnas.171319598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bokar J. A., Shambaugh M. E., Polayes D., Matera A. G., Rottman F. M. Purification and cDNA cloning of the AdoMet-binding subunit of the human mRNA (N6-adenosine)-methyltransferase. RNA. 1997 Nov;3(11):1233–1247. [PMC free article] [PubMed] [Google Scholar]
  6. Cai Z., Tinoco I., Jr Solution structure of loop A from the hairpin ribozyme from tobacco ringspot virus satellite. Biochemistry. 1996 May 14;35(19):6026–6036. doi: 10.1021/bi952985g. [DOI] [PubMed] [Google Scholar]
  7. Connell G. J., Yarus M. RNAs with dual specificity and dual RNAs with similar specificity. Science. 1994 May 20;264(5162):1137–1141. doi: 10.1126/science.7513905. [DOI] [PubMed] [Google Scholar]
  8. Fahnestock S., Neumann H., Shashoua V., Rich A. Ribosome-catalyzed ester formation. Biochemistry. 1970 Jun 9;9(12):2477–2483. doi: 10.1021/bi00814a013. [DOI] [PubMed] [Google Scholar]
  9. Green R., Noller H. F. Ribosomes and translation. Annu Rev Biochem. 1997;66:679–716. doi: 10.1146/annurev.biochem.66.1.679. [DOI] [PubMed] [Google Scholar]
  10. Gutell R. R., Cannone J. J., Shang Z., Du Y., Serra M. J. A story: unpaired adenosine bases in ribosomal RNAs. J Mol Biol. 2000 Dec 1;304(3):335–354. doi: 10.1006/jmbi.2000.4172. [DOI] [PubMed] [Google Scholar]
  11. Gutell R. R., Gray M. W., Schnare M. N. A compilation of large subunit (23S and 23S-like) ribosomal RNA structures: 1993. Nucleic Acids Res. 1993 Jul 1;21(13):3055–3074. doi: 10.1093/nar/21.13.3055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. LAWLEY P. D., BROOKES P. FURTHER STUDIES ON THE ALKYLATION OF NUCLEIC ACIDS AND THEIR CONSTITUENT NUCLEOTIDES. Biochem J. 1963 Oct;89:127–138. doi: 10.1042/bj0890127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lawley P. D., Shah S. A. Methylation of ribonucleic acid by the carcinogens dimethyl sulphate, N-methyl-N-nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine. Comparisons of chemical analyses at the nucleoside and base levels. Biochem J. 1972 Jun;128(1):117–132. doi: 10.1042/bj1280117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Macon J. B., Wolfenden R. 1-Methyladenosine. Dimroth rearrangement and reversible reduction. Biochemistry. 1968 Oct;7(10):3453–3458. doi: 10.1021/bi00850a021. [DOI] [PubMed] [Google Scholar]
  15. Maden B. E., Monro R. E. Ribosome-catalyzed peptidyl transfer. Effects of cations and pH value. Eur J Biochem. 1968 Nov;6(2):309–316. doi: 10.1111/j.1432-1033.1968.tb00450.x. [DOI] [PubMed] [Google Scholar]
  16. Miskin R., Zamir A., Elson D. Inactivation and reactivation of ribosomal subunits: the peptidyl transferase activity of the 50 s subunit of Escherihia coli. J Mol Biol. 1970 Dec 14;54(2):355–378. doi: 10.1016/0022-2836(70)90435-3. [DOI] [PubMed] [Google Scholar]
  17. Muth G. W., Ortoleva-Donnelly L., Strobel S. A. A single adenosine with a neutral pKa in the ribosomal peptidyl transferase center. Science. 2000 Aug 11;289(5481):947–950. doi: 10.1126/science.289.5481.947. [DOI] [PubMed] [Google Scholar]
  18. Nissen P., Hansen J., Ban N., Moore P. B., Steitz T. A. The structural basis of ribosome activity in peptide bond synthesis. Science. 2000 Aug 11;289(5481):920–930. doi: 10.1126/science.289.5481.920. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Ortoleva-Donnelly L., Szewczak A. A., Gutell R. R., Strobel S. A. The chemical basis of adenosine conservation throughout the Tetrahymena ribozyme. RNA. 1998 May;4(5):498–519. doi: 10.1017/s1355838298980086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pestka S. Peptidyl-puromycin synthesis on polyribosomes from Escherichia coli. Proc Natl Acad Sci U S A. 1972 Mar;69(3):624–628. doi: 10.1073/pnas.69.3.624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Polacek N., Gaynor M., Yassin A., Mankin A. S. Ribosomal peptidyl transferase can withstand mutations at the putative catalytic nucleotide. Nature. 2001 May 24;411(6836):498–501. doi: 10.1038/35078113. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Ryder S. P., Ortoleva-Donnelly L., Kosek A. B., Strobel S. A. Chemical probing of RNA by nucleotide analog interference mapping. Methods Enzymol. 2000;317:92–109. doi: 10.1016/s0076-6879(00)17008-9. [DOI] [PubMed] [Google Scholar]
  25. Seela F., Debelak H., Usman N., Burgin A., Beigelman L. 1-Deazaadenosine: synthesis and activity of base-modified hammerhead ribozymes. Nucleic Acids Res. 1998 Feb 15;26(4):1010–1018. doi: 10.1093/nar/26.4.1010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Singer B., Fraenkel-Conrat H. Chemical modification of viral ribonucleic acid. 8. The chemical and biological effects of methylating agents and nitrosoguanidine on tobacco mosaic virus. Biochemistry. 1969 Aug;8(8):3266–3269. doi: 10.1021/bi00836a020. [DOI] [PubMed] [Google Scholar]
  27. Singer B., Fraenkel-Conrat H. Chemical modification of viral ribonucleic acid. VII. The action of methylating agents and nitrosoguanidine on polynucleotides including tobacco mosaic virus ribonucleic acid. Biochemistry. 1969 Aug;8(8):3260–3266. doi: 10.1021/bi00836a019. [DOI] [PubMed] [Google Scholar]
  28. Stern S., Moazed D., Noller H. F. Structural analysis of RNA using chemical and enzymatic probing monitored by primer extension. Methods Enzymol. 1988;164:481–489. doi: 10.1016/s0076-6879(88)64064-x. [DOI] [PubMed] [Google Scholar]
  29. Strobel S. A., Shetty K. Defining the chemical groups essential for Tetrahymena group I intron function by nucleotide analog interference mapping. Proc Natl Acad Sci U S A. 1997 Apr 1;94(7):2903–2908. doi: 10.1073/pnas.94.7.2903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Thompson J., Kim D. F., O'Connor M., Lieberman K. R., Bayfield M. A., Gregory S. T., Green R., Noller H. F., Dahlberg A. E. Analysis of mutations at residues A2451 and G2447 of 23S rRNA in the peptidyltransferase active site of the 50S ribosomal subunit. Proc Natl Acad Sci U S A. 2001 Jul 24;98(16):9002–9007. doi: 10.1073/pnas.151257098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Warner J. R., Gorenstein C. The ribosomal proteins of Saccharomyces cerevisiae. Methods Cell Biol. 1978;20:45–60. doi: 10.1016/s0091-679x(08)62008-7. [DOI] [PubMed] [Google Scholar]
  32. Welch M., Chastang J., Yarus M. An inhibitor of ribosomal peptidyl transferase using transition-state analogy. Biochemistry. 1995 Jan 17;34(2):385–390. doi: 10.1021/bi00002a001. [DOI] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES