1618–1627 Nucleic Acids Research, 2005, Vol. 33, No. 5 doi:10.1093/nar/gki308 Chemical engineering of the peptidyl transferase center reveals an important role of the 20-hydroxyl group of A2451 1 2 Matthias D. Erlacher, Kathrin Lang , Nisha Shankaran , Brigitte Wotzel, Alexander Hu¨ ttenhofer, Ronald Micura1, Alexander S. Mankin2 and Norbert Polacek* Innsbruck Biocenter, Division of Genomics and RNomics—Innsbruck Medical University, Fritz-Pregl Strasse 3, 6020 Innsbruck, Austria, 1Center for Molecular Biosciences Innsbruck (CMBI), Institute of Organic Chemistry, Leopold Franzens University, Innrain 52a, 6020 Innsbruck, Austria and 2Center for Pharmaceutical Biotechnology—M/C 870, University of Illinois at Chicago, 900 S. Ashland Avenue, Chicago, IL 60607, USA Received January 6, 2005; Revised and Accepted February 28, 2005 ABSTRACT The main enzymatic reaction of the large ribosomal subunit is peptide bond formation. Ribosome crystal- lography showed that A2451 of 23S rRNA makes the closest approach to the attacking amino group of aminoacyl-tRNA. Mutations of A2451 had relatively small effects on transpeptidation and failed to unequi- vocally identify the crucial functional group(s). Here, we employed an in vitro reconstitution system for chemical engineering the peptidyl transferase center by introducing non-natural nucleosides at position A2451. This allowed us to investigate the peptidyl transfer reaction performed by a ribosome that contained a modified nucleoside at the active site. The main finding is that ribosomes carrying a 20-deoxyribose at A2451 showed a compromised pep- tidyl transferase activity. In variance, adenine base modifications and even the removal of the entire nuc- leobase at A2451 had only little impact on peptide bond formation, as long as the 20-hydroxyl was pre- sent. This implicates a functional or structural role of the 20-hydroxyl group at A2451 for transpeptidation. INTRODUCTION The ribosome, a multifunctional ribonucleo-protein complex, assembles polypeptides according to the genetic information encoded by mRNAs. Functions of the ribosome in protein synthesis are complex and involve different types of activities critical for decoding of the genetic information, assembling of polypeptide chains, and the proper targeting and release of the synthesized protein. However, there are only two chemical reactions crucial for protein synthesis involving formation or breaking of covalent bonds that are directly catalyzed by the ribosome: peptide bond formation and peptide release of the nascent polypeptide from tRNA. Both of these reactions take place in the active site of the ribosomal peptidyl trans- ferase center (PTC)—a catalytic center located in the large ribosomal subunit. The recent advancement in ribosome crystallography provided a detailed view of the structural organization of the peptidyl transferase active site and conﬁrmed the notion that the PTC is an RNA enzyme (1,2). Investigation of com- plexes of the Haloarcula marismortui large ribosomal subunit with analogs of the peptidyl transferase substrates showed that the cavity of the PTC, which accommodates donor (peptidyl- tRNA) and acceptor (aminoacyl-tRNA) substrates of the pep- tidyl transfer reaction, is decorated by several conserved 23S rRNA residues. These residues are therefore of potential func- tional signiﬁcance and can be involved in substrate positioning and/or chemical catalysis of transpeptidation. The close prox- imity of the N3 of A2451 to the putative position of the tetra- hedral carbon center of the transition state intermediate drew a lot of attention to this nucleotide, and made the nucleobase of this residue one of the most ‘suspicious’ in 23S rRNA for directly participating in peptide bond formation (1). Despite the structural identiﬁcation of the potential molecu- lar players involved in forging a peptide bond, the detailed mechanism of catalysis remains controversial and far from being understood (3–5). Mutational studies failed to unequi- vocally identify functional groups crucial for peptide bond formation. Base substitutions at several conserved nucleotides in the PTC active site, including A2451, had relatively minor effects on the peptidyl transfer reaction (6–10). Although somewhat different on a quantitative scale, all mutational *To whom correspondence should be addressed. Tel: +43 512 507 3384; Fax: +43 512 507 9880; Email: norbert.polacek@uibk.ac.at ª The Author 2005. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oupjournals.org