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

Daniel Herschlag, Fritz Eckstein, Thomas R. Cech
1993 Biochemistry  
The ribozyme derived from the intervening sequence of Tetrahymena thermophila pre-rRNA catalyzes a site-specific endonuclease reaction with both R N A and DNA oligonucleotides: CCCUC-UAAAAA + G + CCCUCU + GAAAAA. However, the R N A substrate (rS) binds -104-fold stronger than the DNA substrate (dS) and once bound reacts -104-fold faster. Here we have investigated the role of individual 2'-hydroxyl groups by comparing the binding and reactivity of "chimeric" oligonucleotide substrates, in which
more » ... he 2'-substituents of the individual sugar residues have been varied. Chimeric substrates containing a single ribonucleotide at positions -6 to +3 (numbered from the cleavage site) were cleaved faster thandS byfactorsof 3.5,3.5,2.3,65,18,1700,7.8,1.7, and 1.4 [(kcat/Km)chimcfic /<kat/Km)dSl. The sum of the energetic contributions from the individual 2'-hydroxyl groups of 13.3 kcal/mol accounts for the 12.2 kcal/mol greater stabilization for R N A than for DNA in binding 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 interactions, rather than overall helical differences, largely account for the different binding and reactivity of the DNA and R N A substrates. Each 2'-hydroxyl group was evaluated for its effect on each of three reaction steps leading to the chemical transition state: two binding steps (duplex formation and docking into tertiary interactions) and the chemical cleavage step. The 2'-hydroxyl groups at positions -3 and -2 stabilize docking, and this stabilization is maintained in the chemical step. This "uniform binding" indicates that these interactions contribute to catalysis by positioning the oligonucleotide substrate for reaction. The 2'-hydroxyl at position +1 has a small effect on the binding 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 exerts its large effect nearly exclusively on the chemical step [Herschlag, D., Eckstein, F., & Cech, T. R. (1993) Biochemistry (following paper in this issue)]. The energetic effects of other modifications of the 2'-substituents provide a crude picture of the active site. The 2'-OCH3 substituent at position-3 inhibits the reaction -10-fold relative to 2'-H, suggesting that an unfavorable interaction cannot be avoided by an isoenergetic structural rearrangement. Furthermore, this binding pocket of the ribozyme has a high degree of specificity: 2'-F, -NH2, and -NH3+ are also ineffective substitutes for the 2'-OH moiety at position -3, even though these substituents lack the steric bulk of the O-methyl group. These effects suggest that this binding site composed of R N A has some rigidity and can discriminate between substrates at the level of single functional groups. The RNA enzyme or "ribozyme" derived from the selfsplicing intron of Tetrahymena thermophila pre-rRNA catalyzes a site-specificendonuclease reaction with both RNA and DNA oligonucleotides (S has a sequence analogous to that adjoining the 5' splice site of the pre-rRNA and is recognized by base pairing to the 5' -5 -3 -1 +2 exon binding site of the ribozyme to form the P 1 duplex (Figure 1 ). Even though the RNA and DNA oligonucleotides can form the same base pairs in the P1 duplex, the DNA substrate binds to the ribozyme -104-fold weaker than the RNA substrate and once bound reacts -IO4-fold slower (Herschlag & Cech, 1990a,c). This corresponds to a difference of 12.2 kcal/mol in total transition-state stabilization (1 0 mM MgCl2, 50 "C). There are two limiting models for this difference: (1) localized interactions that involve one or more of the 2'hydroxyl groups of the RNA substrate are missing with the DNA substrate; or (2) overall geometrical differences between the duplex containing the RNA oligonucleotide substrate and the 5' exon binding site of the ribozyme (Figure 1) and that containing the DNA oligonucleotide allow the RNA substrate to fit into the active site better than the DNA substrate. The observations that RNA oligonucleotides bind -1 04-fold stronger than predicted for simple base pairing interactions with the 5' exon binding site and that DNA oligonucleotides bind about as strongly as predicted for simple duplexes 0
doi:10.1021/bi00083a034 pmid:7688572 fatcat:4qorhnvkmrasxod74gwjg7vpru