Participation of the 3'-CCA of tRNA in the Binding of Catalytic Mg2+ Ions by Ribonuclease P

Biochemistry, 37 (20), 7277 -7283, 1998. bi973100z S0006-2960(97)03100-0
Web Release Date: April 30, 1998

Participation of the 3'-CCA of tRNA in the Binding of Catalytic Mg²⁺ Ions by Ribonuclease P

Bong-Kyeong Oh, Daniel N. Frank, and Norman R. Pace*

Departments of Chemistry and Biology, Indiana University, Bloomington, Indiana 47405, Graduate School of Biotechnology, Korea University, Anam-dong 5ga-1, Sungbuk-ku, Seoul, 136-701, South Korea, and Departments of Plant and Microbial Biology and Molecular and Cell Biology, 111 Koshland Hall, University of California, Berkeley, California 94720-3102

Received December 17, 1997

Revised Manuscript Received March 20, 1998

Abstract:

Ribonuclease P (RNase P) contains a catalytic RNA that cleaves precursor tRNA (pre-tRNA) to form the mature 5'-end of tRNA. Previous kinetic analyses with mutant pre-tRNAs indicated that both C residues of the invariant 3'-terminal CCA form specific interactions with RNase P RNA that contribute to the energetics of substrate binding (1, 2). In the present study, we have used single-turnover kinetic analysis to investigate whether specific changes in the 3'-terminal CCA influence the rate of the chemical step through which enzyme-bound substrate is converted to product (k₂). At optimal ionic strength (1.0 M NH₄Cl, 25 mM MgCl₂), deletion or substitution of the 3'-proximal C residue (CCA) reduced the rate of the chemical step of cleavage (k₂) by 60-fold. Similar changes to the 5'-proximal C residue (CCA) or the 3'-terminal A residue (CCA) reduced k₂ only a few fold. Each mutant substrate exhibited weakened affinity for Mg²⁺, as measured by Hill plots, and the severity of these defects correlated with the observed reductions in k₂. Furthermore, elevated concentrations of Mg²⁺ partially, but not completely, suppress the k₂ defects caused by deletion or substitution of the 3'-proximal C residue. We conclude that the 3'-CCA of pre-tRNA, particularly the 3'-proximal C residue, comprises part of the catalytic pocket formed in the pre-tRNA-RNase P complex and participates in the binding of Mg²⁺ ions that are essential for catalysis by RNase P RNA.

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