Nucleic Acids Research, 2003, Vol. 31, No. 22 6536-6542
© 2003 Oxford University Press
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Mononucleotide derivatives as ribosomal P-site substrates reveal an important contribution of the 2'-OH to activity
Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, Institut für Med. Biochemie, Universität Wien, Dr. Bohrgasse 9/3, A-1030 Wien, Austria and 1 Institut 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{at}bch.univie.ac.at
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
The chemical synthesis of various acylaminoacylated mononucleotides is described and their activities as donor substrates for the ribosomal peptide synthesis were investigated using PhetRNAPhe as an acceptor. This minimal reaction was characterized in detail and was shown to be stimulated by CMP, cytidine and cytosine. By using several cytidine and cytosine analogs evidence is provided that this enhancement is rather caused by base pairing to rRNA, followed by a structural change, than by a base mediated general acid/base catalysis. Only derivatives of AMP proved active as P-site substrates. Further, a significant contribution of the 2'-OH to activity was indicated by the finding that AcLeu-dAMP was inactive as donor substrate, although it is a good inhibitor of peptide bond formation and thus, is presumably bound to the P-site. However, Di(AcLeu)-2'-OCH3-Ade and DiAcLeu-AMP were moderately active in this assay suggesting that the reactivity of the 3'-acylaminoacid ester is stimulated by the presence of the 2'-oxygen group. A model is discussed how further interactions of the 2'-OH in the transition state might influence peptidyl transferase activity.
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