Contributions of 2'-hydroxyl groups of the RNA substrate to binding

Biochemistry 1993,32. 8299-83 11

8299

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,’J Fritz Eckstein,l and Thomas R. Cechll Department of Biochemistry, 8400 Beckman Center, Stanford University, Stanford, California 94305-5307, Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, and Max- Planck- Institut fuer experimentelle Medizin, Hermann-Rein Strasse 3, W-3400 Gottingen, Germany Received February 1I , 1993; Revised Manuscript Received May 27, I993

ABSTRACT: 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: CCCUCUAAAAA G + CCCUCU GAAAAA. However, the R N A substrate (rS) binds 104-foldstronger than the DNA substrate (dS) and once bound reacts 104-foldfaster. Here we have investigated the role of individual 2’-hydroxyl groups by comparing the binding and reactivity of “chimeric” oligonucleotide substrates, in which the 2’-substituents of the individual sugar residues have been varied. Chimeric substrates containing a single ribonucleotide a t 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 /