Sequence-specific endoribonuclease activity of the Tetrahymena ribozyme: enhanced cleavage of certain oligonucleotide substrates that form mismatched ribozyme-substrate complexes.
A shortened form of the self-splicing intervening sequence RNA of Tetrahymena acts as a sequence-specific endoribonuclease. Specificity of cleavage is determined by Watson-Crick base pairing between the active site of the RNA enzyme (ribozyme) and its RNA substrate [Zaug, A. J., Been, M. D., & Cech, T. R. (1986) Nature (London) 324, 429-433]. Surprisingly, single-base changes in the substrate RNA 3 nucleotides preceding the cleavage site, giving a mismatched substrate-ribozyme complex, enhance the rate of cleavage. Mismatched substrates show up to a 100-fold increase in kcat and, in some cases, in kcat/Km. A mismatch introduced by changing a nucleotide in the active site of the ribozyme has a similar effect. Addition of 2.5 M urea or 3.8 M formamide or decreasing the divalent metal ion concentration from 10 to 2 mM reverses the substrate specificity, allowing the ribozyme to discriminate against the mismatched substrate. The effect of urea is to decrease kcat and kcat/Km for cleavage of the mismatched substrate; Km is not significantly affected at 0-2.5 M urea. Thus, progressive destabilization of ribozyme-substrate pairing by mismatches or by addition of a denaturant such as urea first increases the rate of cleavage to an optimum value and then decreases the rate.
Zaug
A J
AJ
Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Boulder 80309-0215.
Grosshans
C A
CA
Cech
T R
TR
D000818:Animals; D001483:Base Sequence; D002122:Calcium Chloride / Q000494:pharmacology; D002413:Cations, Divalent; D004722:Endoribonucleases / Q000378:metabolism*; D005559:Formamides / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D009843:Oligoribonucleotides / Q000378:metabolism*; D012322:RNA Precursors; D012326:RNA Splicing; D016337:RNA, Catalytic; D012335:RNA, Ribosomal / Q000378:metabolism*; D013768:Tetrahymena / Q000032:analysis*; D014158:Transcription, Genetic; D014508:Urea / Q000494:pharmacology
D002413:Cations, Divalent; D005559:Formamides; D009843:Oligoribonucleotides; D012322:RNA Precursors; D016337:RNA, Catalytic; D012335:RNA, Ribosomal; D015636:Magnesium Chloride; C031066:formamide; D014508:Urea; D004722:Endoribonucleases; D008274:Magnesium; D002122:Calcium Chloride