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 Seal 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 pK(app) = 6.9 (10 mM MgCl2, 50 degrees C). This result was surprising because the titratable groups of RNA have pK(a) values of < similar to 4 or > similar to 9. Thus, two models were considered: (i) the ribozyme structure perturbs a pK(a) such that the pK(app) of 6.9 corresponds to an actual titration or (ii) the pK(app)is a kinetic pK(a), 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 fate at high pH. Similarly, substitution of the pro-R(P) 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. The variations in the rate caused by these substitutions below pH 6.9 suggest that the chemical cleavage step is rate-limiting at low pH whereas the similar rates above pH 6.9 suggest that a conformational step is rate-limiting at higher pH (model ii above). Further, the pH dependence suggests that a proton is lost from the E.S.G ternary complex prior to the chemical cleavage step. The approaches described herein should be useful in deconvoluting individual steps in more complex reactions such as self-splicing of group I introns and intron excision from pre-mRNA by the spliceosome.