Comparison of pH dependencies of the Tetrahymena ribozyme reactions with RNA 2'-substituted and phosphorothioate substrates reveals a rate-limiting conformational step.
The L-21 ScaI ribozyme (E) derived from the self-splicing group I intron of Tetrahymena pre-rRNA catalyzes an RNA endonuclease reaction analogous to the first step in self-splicing: CCCUCUAAAAA (S) + G-->CCCUCU+GAAAAA. We show herein that the pH dependence for the single-turnover reaction E.S+G-->products follows a pH dependence with pKapp = 6.9 (10 mM MgCl2, 50 degrees C). This result was surprising because the titratable groups of RNA have pKa values of < approximately 4 or > approximately 9. Thus, two models were considered: (i) the ribozyme structure perturbs a pKa such that the pKapp of 6.9 corresponds to an actual titration or (ii) the pKapp is a kinetic pKa, reflecting a change in the rate-limiting step rather than an actual titration. Oligonucleotide substrates with -H (deoxyribose), -F (2'-fluoro-2'-deoxyribose), and -OH (ribose) substitutions at the 2' position of the U residue at the cleavage site [U(-1)] vary considerably in their intrinsic reactivities. In the ribozyme reaction these substrates reacted at very different rates at low pH, but approached the same limiting reaction rate at high pH. Similarly, substitution of the pro-RP nonbridging oxygen atom of the reactive phosphoryl group by sulfur lowers the intrinsic reactivity of the oligonucleotide substrate. In the ribozyme reaction, this "thio effect" was 2.3 below pH 6.9, whereas the thio substitution had no effect on the rate above pH 6.9.(ABSTRACT TRUNCATED AT 250 WORDS)
Herschlag
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D
Department of Biochemistry, Stanford University, California 94305-5307.
Khosla
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M
D000818:Animals; D001483:Base Sequence; D006863:Hydrogen-Ion Concentration; D007438:Introns; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013768:Tetrahymena / Q000201:enzymology* / Q000235:genetics; D013873:Thionucleotides
D009838:Oligodeoxyribonucleotides; D012322:RNA Precursors; D016337:RNA, Catalytic; D013873:Thionucleotides