Catalysis of RNA cleavage by a ribozyme derived from the group I intron of Anabaena pre-tRNA(Leu).
In the cyanobacterium Anabaena PCC7120, the precursor to tRNA(Leu) contains a 249-nucleotide group I intron that undergoes efficient self-splicing in vitro. By deleting the 5' and 3' splice sites, this intron has now been converted to an RNA enzyme that uses a guanosine nucleophile to cleave substrate RNAs (S) with multiple turnover. This Anabaena ribozyme has a second-order rate constant for RNA cleavage (kcat/Km)S that is 250-500-fold smaller than that of the Tetrahymena ribozyme, and a multiple-turnover rate constant at saturating S [kcat(mt)] that is approximately 400-fold larger. Several lines of evidence, including kinetic analysis of cleavage of phosphorothioate- and deoxynucleotide-substituted substrates and pH dependence, support the conclusion that both (kcat/Km)S and kcat(mt) are limited by the actual chemical cleavage step. In contrast, for the Tetrahymena ribozyme, it has been shown that neither of these rate constants reflects the chemical step. These kinetic differences are expected from the shorter guide sequence-substrate pairing of the Anabaena ribozyme; for example, weaker binding of RNA speeds product release during multiple turnover and thereby overcomes the rate-limiting product release observed for the Tetrahymena ribozyme. Thus, the large kinetic differences represent superficial rather than fundamental differences between these ribozymes. Furthermore, the strength of the guanosine-binding interaction, the stereospecificity for Rp-phosphorothioate at the cleavage site, and the 10(3)-fold slower cleavage with a deoxyribonucleoside leaving group are properties conserved between the Anabaena and Tetrahymena ribozymes. Finally, log(kcat/Km)S increases linearly with pH in the acid range where chemistry is rate-limiting and becomes pH-independent above pH 7, perhaps because a conformational step becomes rate-limiting; again, these are characteristics shared with the Tetrahymena ribozyme. We conclude that two group I ribozymes, although differing in the identity of many of their active site nucleotides, nevertheless provide functionally similar active sites for sequence-specific RNA cleavage.
Zaug
A J
AJ
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
Dávila-Aponte
J A
JA
Cech
T R
TR
D017033:Anabaena / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D007438:Introns / Q000502:physiology*; D007700:Kinetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000235:genetics*; D012329:RNA, Bacterial / Q000235:genetics* / Q000378:metabolism*; D016337:RNA, Catalytic / Q000378:metabolism*; D016053:RNA, Protozoan / Q000235:genetics; D012356:RNA, Transfer, Leu / Q000235:genetics*; D013379:Substrate Specificity; D013768:Tetrahymena / Q000235:genetics; D013816:Thermodynamics; D014158:Transcription, Genetic
D012322:RNA Precursors; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D016053:RNA, Protozoan; D012356:RNA, Transfer, Leu; C091001:pre-tRNA(Leu)