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 smallest group I intron (subgroup IC3) that comes from the pre-tRNA(Ile) of the bacterium Azoarcus sp. BH72. Despite the small size of the Azoarcus ribozyme (195 nucleotides (nt)), it binds tightly to the guanosine nucleophile (Kd = 15 +/- 3 microM) and exhibits activity at high temperatures (approximately 60-70 degrees C). These features may be due to the two GA3 tetraloop interactions postulated in the intron and the high GC content of the secondary structure. The second order rate constant for the Azoarcus ribozyme, ((k(cat)/Km)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 Anabaena PCC7120. pH dependence studies and kinetic analyses of deoxy-substituted substrates suggest that the chemical cleavage step is the rate-determining process in the Azoarcus ribozyme. This may be due to the short 3-nt guide sequence-substrate pairing present in the Azoarcus ribozyme. Finally, the Azoarcus ribozyme shares features conserved in other group I ribozymes including the pH profile, the stereospecificity for the Rp-phosphorothioate at the cleavage site and the 1000-fold decrease in cleavage rate with a deoxyribonucleoside leaving group.
Kuo
L Y
LY
Department of Chemistry, Lewis and Clark College, Portland, OR 97219, USA. kuo@lclark.edu
Davidson
L A
LA
Pico
S
S
D020627:Azoarcus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003854:Deoxyribonucleotides / Q000378:metabolism; D006151:Guanosine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D009943:Organophosphorus Compounds / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D003854:Deoxyribonucleotides; D009943:Organophosphorus Compounds; D016337:RNA, Catalytic; D006151:Guanosine