Pre-steady-state and stopped-flow fluorescence analysis of Escherichia coli ribonuclease III: insights into mechanism and conformational changes associated with binding and catalysis

Mechanistic Studies Reveal Similar Catalytic Strategies for Phosphodiester Bond Hydrolysis by Protein-only and RNA-dependent Ribonuclease P

“…A possible alternate mechanism is activation of a metal-bound nucleophile by a general base as suggested for EcoRV and RNase H (45,46). However, two ionizations (slope ϭ 2) and pK a values of ϳ6.0 -7.0 are observed in pH profiles of these enzymes (45)(46)(47), which is significantly different from the pH dependence observed with PRORP1. Thus, it is unlikely that PRORP1 uses a general base mechanism for activation of the nucleophile.…”

Molecular Requirements for Duplex Recognition and Cleavage by Eukaryotic RNase III: Discovery of an RNA-dependent DNA Cleavage Activity of Yeast Rnt1p

“…Indeed, the proposed mechanism of bacterial RNase III hydrolysis involves the deprotonation of water, producing the hydroxide ion nucleophile, and protonation of the 3 0 oxygen atom of the leaving group. 46 Rnt1p catalytic efficiency is not affected by the presence of deoxyribonucleotides near the cleavage site, but is greatly reduced by replacing one complete RNA strand with DNA (DNA -RNA -RNA).…”

A fluorescence correlation spectroscopy-based enzyme assay for human Dicer

“…Application of pre-steady-state and stopped-fl ow analysis of intrinsic fl uorescence, a Ca 2 + -induced stable enzyme-substrate (ES) complex formation, has previously been reported for E. coli RNase III (Campbell et al , 2002 ). The catalytically active RNase III domains of RNase III form an intermolecular homodimer (Blaszczyk et al , 2001 ).…”

“…Hybridisation was performed as described (Kolb et al , 2005 ) with modifi cations: after heating for 3 min at 95 ° C, the sample was cooled down by 1 ° C every 1.2 min each until a temperature of 25 ° C was reached. Campbell et al , 2002 ). Enzymes were stored on ice and added to the substrate to start the reaction.…”