A unique mechanism for RNA catalysis: the role of metal cofactors in hairpin ribozyme cleavage

Abstract

These results provide clear support for a model of RNA catalysis that does not involve direct coordination of magnesium to the phosphate ester, nor activation of a bound water molecule. A mechanism in which catalysis is carried out by functional groups on the RNA ribozyme itself is possible; such functional groups are likely to have pKa values that are appropriate for carrying out this catalysis. The metal cofactor would then serve to define the architecture of the catalytic pocket and contribute to the stabilization of transient species, as has been described earlier. Hydrolytic pathways in nucleic acid reactions are apparently more diverse than was previously thought, and the hairpin ribozyme falls into a mechanistically distinct class from the Tetrahymena and the hammerhead ribozymes.

Comparison of the hammerhead cleavage reactions stimulated by monovalent and divalent cations

“…The hammerhead ribozyme is a self-cleaving RNA motif found in several plant satellite RNA and viroid genomes (Forster & Symons, 1987a, 1987bUhlenbeck, 1987; Fig+ 1)+ It catalyzes an intramolecular phosphoryl transfer reaction to generate two products, one with a 29-39 cyclic phosphate and the other with a 59-hydroxyl+ The hammerhead has been modified to develop an experimentally accessible, kinetically well-defined system in which to study catalysis (e+g+, Uhlenbeck, 1987;Hertel et al+, 1994Hertel et al+, , 1998Stage-Zimmermann & Uhlenbeck, 1998)+ The in vitro reaction is most efficient in millimolar quantities of divalent metal cations and displays a rate order dependency on one metal ion+ These observations are consistent with the view that RNA catalysis might generally involve metal ions (Dahm et al+, 1993;Pan et al+, 1993;Steitz & Steitz, 1993;Yarus, 1993;Pyle, 1996;Narlikar & Herschlag, 1997;Feig & Uhlenbeck, 1999)+ More recent experiments, however, have provided strong evidence that many ribozymes can perform catalysis by mechanisms not involving divalent metal ions+ The hairpin ribozyme can carry out efficient catalysis in the absence of divalent metal ions and in the absence of direct metal ion coordination (Hampel & Cowan, 1997;Nesbitt et al+, 1997;Young et al+, 1997;Fedor, 2000)+ It was shown that the hammerhead and the VS ribozymes are active in the absence of divalent metal ions provided that very high concentrations of monovalent cations are present (Murray et al+, 1998a)+ Several in vitro selection experiments have also successfully obtained RNA and DNA catalysts that operate in the apparent absence of divalent metal ions or their direct coordination (e+g+, Geyer & Sen, 1997;Jayasena & Gold, 1997;Roth & Breaker, 1998;Suga et al+, 1998)+ The X-ray crystal structure of the hepatitis delta ribozyme revealed a conserved base held at the position of strand cleavage (Ferre-D'Amare et al+, 1998), and subsequent functional studies provided the first mechanistic evidence for general acid-base catalysis mediated by an RNA base, a protonated C residue (Perrotta et al+, 1999;…”

Mg2+-independent hairpin ribozyme catalysis in hydrated RNA films

“…Ribozymes promote site-specific transesterification cleavage of the phosphodiester backbone of RNA in the absence of protein cofactors (Long & Uhlenbeck, 1993;Burke, 1994)+ Although hammerhead and hairpin ribozymes catalyze analogous reactions and are found in the opposite strands of the satellite RNA of tobacco ringspot virus (sTRSV; Symons, 1994), they have distinct structures and catalytic requirements+ The hairpin ribozyme (Burke et al+, 1996;Earnshaw & Gait, 1997) catalyzes reversible, site-specific cleavage of RNA and the reaction is strongly stimulated by physiological concentrations of divalent cations (Chowrira et al+, 1993)+ Ribozyme activity requires formation of a catalytically proficient structure that positions functional groups and/or metal ions in the proper orientation with respect to the scissile phosphodiester bond+ The metal ions may promote proper folding of the RNA or directly participate in chemical catalysis (Cech, 1990;Dahm & Uhlenbeck, 1991;Pyle, 1993)+ Ribozymes have generally been considered to be metalloenzymes, but it has proven difficult to distinguish structural from catalytic roles for the metal ions+ Recent evidence suggests that the hairpin ribozyme's requirement for cations may be different from that of the other ribozymes, in that (1) a slow spermidine-dependent reaction is observed in the absence of divalent cations (Forster et al+, 1987;Chowrira et al+, 1993;Dahm et al+, 1993) and (2) a robust reaction is supported by cobalt (III) hexammine, indicating that inner-sphere coordination of metal ions is not essential for cleavage (Hampel & Cowan, 1997;Nesbitt et al+, 1997;Young et al+, 1997)+ In dimeric (ϩ) sTRSV RNA, hammerhead selfcleavage reactions occurred in spermidine, spermine, or EDTA to extents comparable to those observed in 5-20 mM Mg 2ϩ (Prody et al+, 1986)+ Recent results have shown that hairpin, hammerhead, and Neurospora VS ribozymes do not require divalent metal ions for either folding or catalysis; molar concentrations of monovalent cations (Na ϩ , Li ϩ , NH 4 ϩ ) were shown to be sufficient for efficient catalysis (Murray et al+, 1998;Nesbitt et al+, 1999)+ Divalent metal-independent nucleic acid enzymes have also been isolated from random sequence pools (Geyer & Sen, 1997;Suga et al+, 1998)+ Thus, catalysis by metal ions is not an essential feature of ribozyme catalysis+…”

NMR study on the impact of metal ion binding and deoxynucleotide substitution upon local structure and stability of a small ribozyme

“…Indeed, the question arises whether the two can be separated at all for such a small ribooligonucleotide. Recent studies of the hairpin ribozyme [34][35][36] suggest that the need for metal ions in the catalytic step could be less important than assumed previously, and that the major role of the bound ions is to create a suitable environment, which does not necessarily imply that this structure must be a rigid one. Rather, a whole set or ensemble of energetically similar, but conformationally different substructures could be present.…”