Characterization of the Azoarcus ribozyme: tight binding to guanosine and substrate by an unusually small group I ribozyme

We report novel chemical properties of the ribozyme derived from the smalle st group I intron (subgroup IC3) that comes from the pre-tRNA(Ile) of the b acterium Azoarcus sp. BH72. Despite the small size of the Azoarcus ribozyme (195 nucleotides (nt)), it binds tightly to the guanosine nucleophile (K-d = 15 +/- 3 mu M) and exhibits activity at high temperatures (similar to 60 -70 degrees C). These features may be due to the two GA(3) tetraloop intera ctions postulated in the intron and the high GC content of the secondary st ructure. The second order rate constant for the Azoarcus ribozyme, ((k(cat) /K-m)(S) = 8.4 +/- 2.1 x 10(-5) M-1 min(-1)) is close to that found for the related ribozyme derived from the pre-tRNA(Ile) of the cyanobacterium Anab aena PCC7120. pH dependence studies and kinetic analyses of deoxy-substitut ed substrates suggest that the chemical cleavage step is the rate-determini ng process in:the Azoarcus ribozyme. This may be due to the short 3-nt guid e sequence-substrate pairing present in the Azoarcus ribozyme. Finally, the Azoarcus ribozyme shares features conserved in other group I ribozymes inc luding the pH profile, the stereospecificity for the Rp-phosphorothioate at the cleavage site and the 1000-fold decrease in cleavage rate with a deoxy ribonucleoside leaving group. (C) 1999 Elsevier Science B.V. All rights res erved.