GENERALITY OF THE NUX RULE - KINETIC-ANALYSIS OF THE RESULTS OF SYSTEMATIC MUTATIONS IN THE TRINUCLEOTIDE AT THE CLEAVAGE SITE OF HAMMERHEAD RIBOZYMES

In order to study in detail the generality of the NUX (N = A, U, G, or C; X = A, U, or C) rule for the GUC triplet adjacent to the cleavage site in hammerhead ribozymes, two kinetic parameters, namely, k(cat) and K-m, were determined for substrates with mutations in this triplet, which included double mutants with mutations of both N and X. All substrates with mutated cleavage sites were cleaved with reduced efficiency compared to the wild type. However, some mutations mainly affected k(cat) and others mainly affected K-m, a phenomenon that could not have been predicted from previous results. A as the first or third base increased K-m by 35- or 30-fold, respectively, while the effect on k(cat) was small. U as the First or third base decreased k(cat) by 8- or 15-fold, respectively, while the effect on K-m was small. The effect of C as the first base on kinetic parameters was relatively small. The kinetic parameters of double mutants generally were determined by the effects of both individual point mutations. The AUA triplet gave a very much higher k(cat) than the other double mutants tested. In general, all of the mutants except for the mutant substrate with the CUC triplet had very low cleavage efficiencies, which ranged from 0.6% to 8% of the wild-type value, as a result of the deleterious effects of the mutations on k(cat), K-m, or both. This kind of analysis can explain why nature has chosen triplets such as GUC, GUA, and AUA in cis reactions: all of these triplets retain high k(cat) values, although the K-m values of GUA and AUA are high.