Evidence for proton transfer in the rate-limiting step of a fast-cleaving Varkud satellite ribozyme.
A fast-cleaving version of the Varkud satellite ribozyme, called RG, shows an apparent cis-cleavage rate constant of 5 sec(-1), similar to the rates of protein enzymes that catalyze similar reactions. Here, we describe mutational, pH-rate, and kinetic solvent isotope experiments that investigate the identity and rate constant of the rate-limiting step in this reaction. Self-cleavage of RG exhibits a bell-shaped rate vs. pH profile with apparent pK(a)s of 5.8 and 8.3, consistent with the protonation state of two nucleotides being important for the rate of cleavage. Cleavage experiments in heavy water (D(2)O) revealed a kinetic solvent isotope effect consistent with proton transfer in the rate-limiting step. A mutant RNA that disrupts a peripheral loop-loop interaction involved in RNA folding exhibits pH- and D(2)O-independent cleavage approximately 10(3)-fold slower than wild type, suggesting that this mutant is limited by a different step than wild type. Substitution of adenosine 756 in the putative active-site loop with cytosine also decreases the cleavage rate approximately 10(3)-fold, but the A756C mutant retains pH- and D(2)O-sensitivity similar to wild type, consistent with this mutant and wild type being limited by the chemical step of the reaction. These results suggest that the RG ribozyme provides a good experimental system to investigate the nature of fast, rate-limiting steps in a ribozyme cleavage reaction.
Smith
M Duane
MD
Department of Molecular and Medical Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8.
Collins
Richard A
RA
D001483:Base Sequence; D002021:Buffers; D017666:Deuterium Oxide / Q000494:pharmacology; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008954:Models, Biological; D009154:Mutation; D009491:Neurospora / Q000201:enzymology; D009690:Nucleic Acid Conformation; D011522:Protons; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism*; D012997:Solvents; D013696:Temperature
D002021:Buffers; D011522:Protons; D016337:RNA, Catalytic; D012331:RNA, Fungal; D012997:Solvents; D004722:Endoribonucleases; C438195:varkud satellite ribozyme; D017666:Deuterium Oxide