PARTICIPATION OF THE 3'-CCA OF TRANSFER-RNA IN THE BINDING OF CATALYTIC MG2-P( IONS BY RIBONUCLEASE)

Ribonuclease P (RNase P) contains a catalytic RNA that cleaves precurs or 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 invest igate whether specific changes in the 3'-terminal CCA influence the ra te of the chemical step through which enzyme-bound substrate is conver ted to product(k(2)). At optimal ionic strength (1.0 M NH4Cl, 25 mM Mg Cl2), deletion or substitution of the 3'-proximal C residue (CCA) redu ced the rate of the chemical step of cleavage (k(2)) by 60-fold. Simil ar changes to the 5'-proximal C residue (CCA) or the 3'-terminal A res idue (CCA) reduced k(2) only a few fold. Each mutant substrate exhibit ed weakened affinity for Mg2+, as measured by Hill plots, and the seve rity of these defects correlated with the observed reductions in k(2). Furthermore, elevated concentrations of Mg2+ partially, but not compl etely, suppress the k(2) defects caused by deletion or substitution of the 3'-proximal C residue. We conclude that the 3'-CCA of pre-tRNA, p articularly 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.