CONTRIBUTIONS OF 2'-HYDROXYL GROUPS OF THE RNA SUBSTRATE TO BINDING AND CATALYSIS BY THE TETRAHYMENA RIBOZYME - AN ENERGETIC PICTURE OF AN ACTIVE-SITE COMPOSED OF RNA

The ribozyme derived from the intervening sequence of Tetrahymena ther mophila pre-rRNA catalyzes a site-specific endonuclease reaction with both RNA and DNA oligonucleotides: CCCUCUAAAAA + G half arrow right ov er half arrow left CCCUCU + GAAAAA. However, the RNA substrate (rS) bi nds approximately 10(4)-fold stronger than the DNA substrate (dS) and once bound reacts approximately 10(4)-fold faster. Here we have invest igated the role of individual 2'-hydroxyl groups by comparing the bind ing and reactivity of ''chimeric'' oligonucleotide substrates, in whic h the 2'-substituents of the individual sugar residues have been varie d. Chimeric substrates containing a single ribonucleotide at positions -6 to +3 (numbered from the cleavage site) were cleaved faster than d S by factors of 3.5, 3.5, 2.3, 65, 18, 1700, 7.8, 1.7, and 1.4[(k(cat) /K(m))chimeric S/(k(cat)/K(m))dS]. The sum of the energetic contributi ons from the individual 2'-hydroxyl groups of 13.3 kcal/mol accounts f or the 12.2 kcal/mol greater stabilization for RNA than for DNA in bin ding and cleavage (i.e., overall transition-state stabilization). This observation and the significant energetic effects from single ribose substitutions at positions -3 to +1 strongly suggest that local intera ctions, rather than overall helical differences, largely account for t he different binding and reactivity of the DNA and RNA substrates. Eac h 2'-hydroxyl group was evaluated for its effect on each of three reac tion steps leading to the chemical transition state: two binding steps (duplex formation and docking into tertiary interactions) and the che mical cleavage step. The 2'-hydroxyl groups at positions -3 and -2 sta bilize docking, and this stabilization is maintained in the chemical s tep. This ''uniform binding'' indicates that these interactions contri bute to catalysis by positioning the oligonucleotide substrate for rea ction. The 2'-hydroxyl at position +1 has a small effect on the bindin g step and an additional small but significant effect on the chemical step. Thus, the ribozyme, like protein enzymes, can take advantage of interactions away from the site of chemistry to provide stabilization specifically in the transition state. The 2'-hydroxyl at position -1 e xerts its large effect nearly exclusively on the chemical step [Hersch lag, D., Eckstein, F., & Cech, T. R. (1993) Biochemistry (following pa per in this issue)]. The energetic effects of other modifications of t he 2'-substituents provide a crude picture of the active site. The 2'- OCH3 substituent at position -3 inhibits the reaction approximately 10 -fold relative to 2'-H, suggesting that an unfavorable interaction can not be avoided by an isoenergetic structural rearrangement. Furthermor e, this binding pocket of the ribozyme has a high degree of specificit y: 2'-F, -NH2, and -NH3+ are also ineffective substitutes for the 2'-O H moiety at position -3, even though these substituents lack the steri c bulk of the O-methyl group. These effects suggest that this binding site composed of RNA has some rigidity and can discriminate between su bstrates at the level of single functional groups.