1626 Nucleic Acids Research, 2005, Vol. 33, No. 5 conformation of the active site, therefore affecting accurate placing of the reaction substrates in the PTC. We would like to note that the gapped-cp-reconstitution inherits some intrinsic limitations of the ‘standard’ in vitro reconstitution systems. One of the major shortcomings is that peptide bond synthesis does not proceed at rates comparable to that found in vivo. Therefore, our system does not allow the measurement of subtle changes in peptidyl transferase activity, but is applicable to pinpoint crucial groups in the PTC whose replacement or removal signiﬁcantly affects the overall rate of transpeptidation, such as the ribose 20-hydroxyl at A2451. In conclusion, we have developed an experimental approach, the gapped-cp-reconstitution of the large ribosomal subunit, which makes it possible to place modiﬁed nucleotides at speciﬁc positions within 23S rRNA. By this approach, we showed that removal of the ribose 20-hydroxyl group at A2451 of 23S rRNA interfered with peptide bond formation. In con- trast, all base modiﬁcations and even the deletion of the entire nucleobase were largely tolerated, as long as the 20-hydroxyl remained in place. Our data suggest that the ribose 20-hydroxyl at position A2451 plays an important role in the catalytic activity of the PTC. ACKNOWLEDGEMENTS We would like to thank S.R. Connell, D. Wilson, T. Tenson and A. Barta for critical reading of the manuscript and valuable suggestions. C. Ho¨ bartner, K. Bakowska and M.J. Gomez are acknowledged for help with experiments. K. Ito and Y. Nakamura are thanked for providing RF1. This work was supported by grants from the Austrian Science Foundation FWF (P16932 to N.P., P15042 and P17864 to R.M.), from the ‘Tiroler Wissenschaftsfonds’ (UNI-404/109 to N.P. and UNI-404/39 to R.M.), and from the National Institutes of Health (GM 59028) to A.S.M. Funding to pay the Open Access publication charges for this article was provided by the FWF grant P16932 (to N.P.). Conflict of interest statement. None declared. REFERENCES 1. Nissen,P., Hansen,J., Ban,N., Moore,P.B. and Steitz,T.A. (2000) The structural basis of ribosome activity in peptide bond synthesis. Science, 289, 920–930. 2. Harms,J., Schluenzen,F., Zarivach,R., Bashan,A., Gat,S., Agmon,I., Bartels,H., Franceschi,F. and Yonath,A. (2001) High resolution structure of the large ribosomal subunit from a mesophilic eubacterium. Cell, 107, 679–688. 3. Barta,A., Dorner,S., Polacek,N., Berg,J.M., Lorsch,J.R., Nissen,P., Hansen,J., Muth,G.W., Ban,N., Moore,P.B. et al. (2001) Mechanism of ribosomal peptide bond formation. Science, 291, 203a. 4. Xiong,L., Polacek,N., Sander,P., Bo¨ttger,E.C. and Mankin,A.S. (2001) pKa of adenine 2451 in the ribosomal peptidyl transferase center remains elusive. RNA, 7, 1365–1369. 5. Bayfield,M.A., Dahlberg,A.E., Schulmeister,U., Dorner,S. and Barta,A. (2001) A conformational change in the ribosomal peptidyl transferase center upon active/inactive transition. Proc. Natl Acad. Sci. USA, 98, 10096–10101. 6. Polacek,N., Gaynor,M., Yassin,A. and Mankin,A.S. (2001) Ribosomal peptidyl transferase can withstand mutations at the putative catalytic nucleotide. Nature, 411, 498–501. 7. Thompson,J., Kim,D.F., O’Connor,M., Lieberman,K.R., Bayfield,M.A., Gregory,S.T., Green,R., Noller,H.F. and Dahlberg,A.E. (2001) Analysis of mutations at residues A2451 and G2447 of 23S rRNA in the peptidyl transferase active site of the 50S ribosomal subunit. Proc. Natl Acad. Sci. USA, 98, 9002–9007. 8. Katunin,V.I., Muth,G.W., Strobel,S.A., Wintermeyer,W. and Rodnina,M.V. (2002) Important contribution to catalysis of peptide bond formation by a single ionizing group within the ribosome. Mol. Cell, 10, 339–346. 9. Polacek,N., Gomez,M.J., Ito,K., Xiong,L., Nakamura,Y. and Mankin,A.S. (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. 10. Youngman,E.M., Brunelle,J.L., Kochaniak,A.B. and 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. 11. Hansen,J.L., Schmeing,T.M., Moore,P.B. and Steitz,T.A. (2002) Structural insights into peptide bond formation. Proc. Natl Acad. Sci. USA, 99, 11670–11675. 12. Khaitovich,P., Tenson,T., Kloss,P. and Mankin,A.S. (1999) Reconstitution of functionally active Thermus aquaticus large ribosomal subunits with in vitro-transcribed rRNA. Biochemistry, 38, 1780–1788. 13. Marcker,K.A. (1965) The formation of N-formyl-methionyl-sRNA. J. Mol. Biol., 14, 63–70. 14. Blaha,G., Stelzl,U., Spahn,C.M., Agrawal,R.K., Frank,J. and Nierhaus,K.H. (2000) Preparation of functional ribosomal complexes and effect of buffer conditions on tRNA positions observed by cryoelectron microscopy. Methods Enzymol., 317, 292–309. 15. Khaitovich,P. and Mankin,A.S. (1999) Effect of antibiotics on large ribosomal subunit assembly reveals possible function of 5S rRNA. J. Mol. Biol., 291, 1025–1034. 16. Ho¨bartner,C., Kreutz,C., Flecker,E., Ottenschla¨ger,E., Pils,W., Grubmayr,K. and Micura,R. (2003) The synthesis of 20-O- [(triisopropylsilyl)-oxy]-methyl (TOM) phosphoramidites of methylated ribonucleosides (m1G, m2G, m22G, m1I, m3U, m4C, m6A, m62A) for use in automated RNA solid-phase synthesis. Monatsh. Chem.—Chemical Monthly, 134, 851–873. 17. Micura,R., Pils,W., Ho¨bartner,C., Grubmayr,K., Ebert,M.-O. and Jaun,B. (2001) Methylation of nucleobases in RNA oligonucleotides mediates hairpin-duplex conversion. Nucleic Acids Res., 29, 3997–4005. 18. Pitsch,S., Weiss,P.A., Jenny,L., Stutz,A. and Wu,X. (2001) Reliable chemical synthesis of oligoribonucleotides (RNA) with 20-O- [(triisopropylsilyl)oxy]methyl(20-O-tom)-protected phosphoramidites. Helv. Chim. Acta, 84, 3773–3795. 19. Micura,R. (2002) Small interfering RNAs and their chemical synthesis. Angew. Chem. Int. Ed. Engl., 41, 2265–2269. 20. Ho¨bartner,C. and Micura,R. (2003) Bistable secondary structures of small RNAs and their structural probing by comparative imino proton NMR spectroscopy. J. Mol. Biol., 325, 421–431. 21. Ito,K. and Nakamura,Y. (1997) Cloning and overexpression of polypeptide release factor 1 of Thermus thermophilus. Biochimie, 79, 287–292. 22. Bashan,A., Agmon,I., Zarivach,R., Schluenzen,F., Harms,J., Berisio,R., Bartels,H., Franceschi,F., Auerbach,T., Hansen,H.A. et al. (2003) Structural basis of the ribosomal machinery for peptide bond formation, translocation, and nascent chain progression. Mol. Cell, 11, 91–102. 23. Kim,D.F. and Green,R. (1999) Base-pairing between 23S rRNA and tRNA in the ribosomal A site. Mol. Cell, 4, 859–864. 24. Bhuta,P., Kumar,G. and Chla´dek,S. (1982) The peptidyltransferase center of Escherichia coli ribosomes: binding sites for the cytidine 30-phosphate residues of the aminoacyl-tRNA 30-terminus and the interrelationship between the acceptor and donor sites. Biochim. Biophys. Acta, 696, 208–211. 25. Noller,H.F. (1984) Structure of ribosomal RNA. Annu. Rev. Biochem., 53, 119–162. 26. Green,R. and Noller,H.F. (1996) In vitro complementation analysis localizes 23S rRNA posttranscriptional modifications that are required for Escherichia coli 50S ribosomal subunit assembly and function. RNA, 2, 1011–1021. 27. Liu,B. and Fournier,J. (2004) Interference probing of rRNA with snoRNPs: a novel approach for functional mapping of RNA in vivo. RNA, 10, 1130–1141. 28. Nierhaus,K.H., Schulze,H. and Cooperman,B.S. (1980) Molecular mechanisms of the ribosomal peptidyl transferase center. Biochem. Int., 1, 185–192.