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Participation of the 3-CCA of tRNA in th ... ytic Mg2 ions by ribonuclease P
Participation of the 3'-CCA of tRNA in the binding of catalytic Mg2+ ions by ribonuclease P.
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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.
Oh BK, Frank DN, Pace NR
Biochemistry
1998-05-19 00:00
37
20
7277-83
Bacillus subtilis,Base Sequence,Binding Sites,Catalysis,Cations, Divalent,Endoribonucleases,Hydrolysis,Kinetics,Magnesium,Molecular Sequence Data,RNA Precursors,RNA, Bacterial,RNA, Catalytic,RNA, Transfer, Asp,Ribonuclease P,Cations, Divalent,RNA Precursors,RNA, Bacterial,RNA, Catalytic,RNA, Transfer, Asp,Magnesium,Endoribonucleases,Ribonuclease P
Department of Chemistry, Indiana University, Bloomington 47405, USA
Biochemistry
NIGMS GM 34527
0006-2960
10.1021/bi973100z
bi973100z
956
True
9585541
