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J. Biol. Chem., Vol. 278, Issue 44, 43394-43401, October 31, 2003

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Catalysis by RNase P RNA

UNIQUE FEATURES AND UNPRECEDENTED ACTIVE SITE PLASTICITY^*

Tina Persson, Simona Cuzic¶, and Roland K. Hartmann¶||

From the Universität zu Lübeck, Institut für Biochemie, Ratzeburger Allee 160, D-23538 Lübeck, Germany

Metal ions are essential cofactors for precursor tRNA (ptRNA) processing by bacterial RNase P. The ribose 2'-OH at nucleotide (nt) –1 of ptRNAs is known to contribute to positioning of catalytic Me²⁺. To investigate the catalytic process, we used ptRNAs with single 2'-deoxy (2'-H), 2'-amino (2'-N), or 2'-fluoro (2'-F) modifications at the cleavage site (nt –1). 2' modifications had small (2.4–7.7-fold) effects on ptRNA binding to E. coli RNase P RNA in the ground state, decreasing substrate affinity in the order 2'-OH > 2'-F > 2'-N > 2'-H. Effects on the rate of the chemical step (about 10-fold for 2'-F, almost 150-fold for 2'-H and 2'-N) were much stronger, and, except for the 2'-N modification, resembled strikingly those observed in the Tetrahymena ribozyme-catalyzed reaction at corresponding position. Mn²⁺ rescued cleavage of the 2'-N but also the 2'-H-modified ptRNA, arguing against a direct metal ion coordination at this location. Miscleavage between nt –1 and –2 was observed for the 2'-N-ptRNA at low pH (further influenced by the base identities at nt –1 and +73), suggesting repulsion of a catalytic metal ion due to protonation of the amino group. Effects caused by the 2'-N modification at nt –1 of the substrate allowed us to substantiate a mechanistic difference in phosphodiester hydrolysis catalyzed by Escherichia coli RNase P RNA and the Tetrahymena ribozyme: a metal ion binds next to the 2' substituent at nt –1 in the reaction catalyzed by RNase P RNA, but not at the corresponding location in the Tetrahymena ribozyme reaction.

Received for publication, June 5, 2003 , and in revised form, August 5, 2003.

^* This work was supported by Deutsche Forschungsgemeinschaft Grant HA 1672/7-3. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Current address: Lund University, Dept. of Chemistry, Organic Chemistry 1, P.O. Box 124, SE-221 00, Lund, Sweden.

^¶ Current address: Philipps-Universität Marburg, Institut für Pharmazeutische Chemie, Marbacher Weg 6, D-35037, Marburg, Germany.

^|| To whom correspondence should be addressed. Tel.: 6421-28-25827; Fax: 6421-28-25854; E-mail: roland.hartmann{at}staff.uni-marburg.de.

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