Metal ion requirements for sequence-specific endoribonuclease activity of the Tetrahymena ribozyme.
A shortened form of the self-splicing intervening sequence RNA of Tetrahymena thermophila acts as an enzyme, catalyzing sequence-specific cleavage of RNA substrates. We have now examined the metal ion requirements of this reaction. Mg2+ and Mn2+ are the only metal ions that by themselves give RNA enzyme activity. Atomic absorption spectroscopy indicates that Zn, Cu, Co, and Fe are not present in amounts equimolar to the RNA enzyme and when added to reaction mixtures do not facilitate cleavage. Thus, these ions can be eliminated as cofactors for the reaction. While Ca2+ has no activity by itself, it alleviates a portion of the Mg2+ requirement; 1 mM Ca2+ reduces the Mg2+ optimum from 2 to 1 mM. These results, combined with studies of the reactivity of mixtures of metal ions, lead us to postulate that two classes of metal ion binding sites are required for catalysis. Class 1 sites have more activity with Mn2+ than with Mg2+, with the other divalent ions and Na+ and K+ having no activity. It is not known if ions located at class 1 sites have specific structural roles or are directly involved in active-site chemistry. Class 2 sites, which are presumably structural, have an order of preference Mg2+ greater than or equal to Ca2+ greater than Mn2+ and Ca2+ greater than Sr2+ greater than Ba2+, with Zn2+, Cu2+, Co2+, Na+, and K+ giving no detectable activity over the concentration range tested.
Grosshans
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Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
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D000818:Animals; D001665:Binding Sites; D002413:Cations, Divalent; D004722:Endoribonucleases / Q000378:metabolism*; D007700:Kinetics; D008670:Metals / Q000494:pharmacology*; D009843:Oligoribonucleotides; D012322:RNA Precursors / Q000378:metabolism*; D012326:RNA Splicing; D016337:RNA, Catalytic; D012335:RNA, Ribosomal / Q000378:metabolism*; D013054:Spectrophotometry, Atomic; D013379:Substrate Specificity; D013768:Tetrahymena / Q000378:metabolism*; D014131:Trace Elements / Q000032:analysis
D002413:Cations, Divalent; D008670:Metals; D009843:Oligoribonucleotides; D012322:RNA Precursors; D016337:RNA, Catalytic; D012335:RNA, Ribosomal; D014131:Trace Elements; D004722:Endoribonucleases