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: 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 the 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 /