Escherichia coli RNase P RNA: substrate ribose modifications at G+1, but not nucleotide -1/+73 base pairing, affect the transition state for cleavage chemistry.

The temperature dependence of processing of precursor tRNA(Gly) (ptRNA(Gly)) variants carrying a single 2'-OCH(3) or locked nucleic acid (LNA) modification at G+1 by Escherichia coli endoribonuclease P RNA was studied at rate-limiting chemistry. We show, for the first time, that these ribose modifications at nucleotide +1 increase the activation energy and alter the activation parameters for the transition state of hydrolysis at the canonical (c(0)) cleavage site (between nucleotides -1 and +1). The modified substrates, particularly the one with LNA at G+1, caused an increase in the activation enthalpy Delta H(double dagger), which was partly compensated for by a simultaneous increase in the activation entropy DeltaS(double dagger).

A 2'-methyl or 2'-methylene group at G+1 in precursor tRNA interferes with Mg2+ binding at the enzyme-substrate interface in E-S complexes of E. coli RNase P.

We analyzed processing of precursor tRNAs carrying a single 2'-deoxy, 2'-OCH(3), or locked nucleic acid (LNA) modification at G+1 by Escherichia coli RNase P RNA in the absence and presence of its protein cofactor. The extra methyl or methylene group caused a substrate binding defect, which was rescued at higher divalent metal ion (M(2+)) concentrations (more efficiently with Mn(2+) than Mg(2+)), and had a minor effect on cleavage chemistry at saturating M(2+) concentrations. The 2'-OCH(3) and LNA modification at G+1 resulted in higher metal ion cooperativity for substrate binding to RNase P RNA without affecting cleavage site selection.

Studies on Escherichia coli RNase P RNA with Zn2+ as the catalytic cofactor.

We demonstrate, for the first time, catalysis by Escherichia coli ribonuclease P (RNase P) RNA with Zn2+ as the sole divalent metal ion cofactor in the presence of ammonium, but not sodium or potassium salts. Hill analysis suggests a role for two or more Zn2+ ions in catalysis. Whereas Zn2+ destabilizes substrate ground state binding to an extent that precludes reliable Kd determination, Co(NH3)6(3+) and Sr2+ in particular, both unable to support catalysis by themselves, promote high-substrate affinity.

Catalysis by RNase P RNA: unique features and unprecedented active site plasticity.

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).