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Nucleic Acids Research 2005 33(5):1618-1627; doi:10.1093/nar/gki308
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Published online 14 March 2005

© 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{at}oupjournals.org


Article

Chemical engineering of the peptidyl transferase center reveals an important role of the 2'-hydroxyl group of A2451

Matthias D. Erlacher, Kathrin Lang1, Nisha Shankaran2, Brigitte Wotzel, Alexander Hüttenhofer, 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 2Center for Pharmaceutical Biotechnology—M/C 870, University of Illinois at Chicago 900 S. Ashland Avenue, Chicago, IL 60607, USA

*To whom correspondence should be addressed. Tel: +43 512 507 3384; Fax: +43 512 507 9880; Email: norbert.polacek{at}uibk.ac.at

Received January 6, 2005. Revised February 28, 2005. Accepted February 28, 2005.

The main enzymatic reaction of the large ribosomal subunit is peptide bond formation. Ribosome crystallography 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 unequivocally 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 2'-deoxyribose at A2451 showed a compromised peptidyl transferase activity. In variance, adenine base modifications and even the removal of the entire nucleobase at A2451 had only little impact on peptide bond formation, as long as the 2'-hydroxyl was present. This implicates a functional or structural role of the 2'-hydroxyl group at A2451 for transpeptidation.


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