PMID:12904300
| Citation |
Persson, T, Cuzic, S and Hartmann, RK (2003) Catalysis by RNase P RNA: unique features and unprecedented active site plasticity. J. Biol. Chem. 278:43394-401 |
|---|---|
| Abstract |
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. |
| Links |
PubMed Online version:10.1074/jbc.M305939200 |
| Keywords |
Animals; Binding Sites; Catalysis; Escherichia coli/enzymology; Escherichia coli/metabolism; Hydrogen-Ion Concentration; Ions; Kinetics; Magnesium/metabolism; Manganese/metabolism; Models, Biological; Nucleic Acid Conformation; Protein Binding; RNA/metabolism; RNA, Catalytic/metabolism; Ribonuclease P/chemistry; Ribonucleases/metabolism; Substrate Specificity; Tetrahymena/metabolism |
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