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Nucleic Acids Research 2005 33(9):3065-3071; doi:10.1093/nar/gki617
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Published online 25 May 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

Activity of 3'-thioAMP derivatives as ribosomal P-site substrates

Silke Dorner, Walther Schmid1 and Andrea Barta*

Medizinische Universität Wien, Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, Department of Biochemistry Dr Bohrgasse 9/3, A-1030 Wien, Austria 1Institut für Org. Chemie, Universität Wien Währingerstrasse 38, A-1090 Wien, Austria

*To whom correspondence should be addressed. Tel: +43 1 4277 61640; Fax: +43 1 4277 9616; Email: Andrea.Barta{at}univie.ac.at

Received February 1, 2005. Revised May 5, 2005. Accepted May 5, 2005.

The ribosome is a large RNP complex but its main enzymatic activity, the peptidyl transferase, is a ribozyme. As many RNA enzymes use divalent metal ions in catalysis, one of the hypotheses put forward proposed that metal ions might aid peptide bond formation. To be able to test a possible coordination of a metal ion to the 3'-bridging oxygen of P-site substrates, a 3'-thioAMP was synthesized. Its chemical acylation with N-acetyl-L-leucine yielded both mono and diaminoacylated 3'-thioAMP. These thioated substrates were tested for peptide bond formation in an optimized fragment reaction in comparison with their unmodified counterparts. As the amino acid was predominantly linked to the unproductive 2'-OH in AcLeu-thioAMP (5), this substrate was barely active and not used for further analysis. In contrast, Di(AcLeu)-thioAMP (4) was more active than Di(AcLeu)-AMP (2) which is in line with the higher energy of thioesters. Both activities were slightly enhanced when Mn2+ containing buffers were employed in the assay. These data show that thioated P-site substrates are active in peptide bond formation and can in principle be used for metal-ion-rescue experiments in a full translation system.

Present address: Silke Dorner, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, 704 PCTB Baltimore, MD 21205, USA


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