An important role of G638 in the cis-cleavage reaction of the Neurospora VS ribozyme revealed by a novel nucleotide analog incorporation method

Abstract

We describe a chemical coupling procedure that allows joining of two RNAs, one of which contains a site-specific base analog substitution, in the absence of divalent ions. This method allows incorporation of nucleotide analogs at specific positions even into large, cis-cleaving ribozymes. Using this method we have studied the effects of substitution of G638 in the cleavage site loop of the VS ribozyme with a variety of purine analogs having different functional groups and pK a values. Cleavage rate versus pH profiles combined with kinetic solvent isotope experiments indicate an important role for G638 in proton transfer during the rate-limiting step of the cis-cleavage reaction.

The ionic environment determines ribozyme cleavage rate by modulation of nucleobase pKa

“…The observed rate in Na + is more than 3000-fold slower than in Mg 2+ , and we find that the apparent pK a values are even further apart (Fig. 1D, <4.5 and >9), in fact, sufficiently far from neutral pH that additional effects observed at extreme pH prevent their accurate measurement (Jaikaran et al 2008; see Materials and Methods). Kinetic solvent isotope experiments confirmed that the reaction in Na + was limited by proton transfer, as in Mg 2+ and Li + (Fig.…”

“…F is the proportion of the RNA capable of participating in catalysis: in the simplest model, this is the proportion of RNA in which the general acid and general base are in their protonated and deprotonated states, f HA + , and f B À , respectively. f HA + and f B À are functions of pH and depend on the pK a values of the general acid and base and the pH at which the reaction is performed (Bevilacqua 2003;Jaikaran et al 2008). We have recently incorporated an additional term into F that describes the cooperative loss of activity at very low pH; this term is a function of pH and the number of sites that contribute to the inactivation of the ribozyme when protonated (Bevilacqua 2003;Jaikaran et al 2008;see Materials and Methods).…”

“…In the VS ribozyme, nucleobases A756 and G638 have been implicated as the most likely candidates for the catalytic nucleobases (Andersen and Collins 2000;Hiley et al 2002;Jones and Strobel 2003;Smith and Collins 2007;Wilson et al 2007;Jaikaran et al 2008). Determining which nucleobase functions as the general acid and which as the general base is challenging for any enzyme because a k obs versus pH curve obtained under a single set of experimental conditions can be equally explained by either of two kinetic models ( Fig.…”

“…Reactions with rate constants >6 min À1 were performed using a Kintek RQF-3 instrument (Kintek Corp.). Buffers, pH measurements, and data quantification were done as previously described (Jaikaran et al 2008). Estimates of k obs in a given reaction condition typically varied by less than 620% in replicate experiments.…”

“…Considering the 3000-fold range in k obs among reactions in Mg 2+ , Li + , and Na + , the small differences in the estimates of k 1 among these ionic conditions are Control experiments showed that the concentrations of cations used were saturating across the pH range examined (see Materials and Methods). Data were fit to either of two kinetically ambiguous models of general acid-base catalysis that also included a term describing the cooperative loss of activity at low pH (Jaikaran et al 2008) (solid lines; see Materials and Methods). In Model 1, the lower pK a is assigned to the general base and the higher pK a to the general acid; these assignments are reversed in Model 2.…”