Four ribose 2'-hydroxyl groups essential for catalytic function of the hairpin ribozyme.
The hairpin ribozyme catalyzes site-specific cleavage of an RNA substrate using a magnesium-dependent transphosphorylation mechanism. Here, we describe experiments designed to test the importance of ribose 2'-hydroxyl groups for ribozyme function. Ribozymes for this work were synthesized in two segments using solid-phase RNA phosphoramidite chemistry. 2'-Deoxyribonucleotides were systematically introduced at each of the 50 positions within the ribozyme, and the catalytic activity of the resulting mixed RNA-DNA polymers was measured. Deletion of the 2'-hydroxyl group at each of four sites (A10, G11, A24, and C25) was found to result in severe inhibition of cleavage activity (kcat/KM decreased by 100- to 1000-fold), although KM measurements and mobility-shift assays showed that substrate binding was not affected. Identical results were obtained upon substitution of these ribonucleotides with 2'-O-methyl derivatives. Inhibition by 2'-modified sugars at G11 or A24 was rescued by increased Mg2+ concentrations, suggesting that these 2'-hydroxyls may function in magnesium binding. Our results demonstrate that the 2'-hydroxyl groups at A10, G11, A24, and C25 provide essential functions for catalysis, possibly forming important tertiary contacts or magnesium coordination sites that are necessary for active site architecture.
Chowrira
B M
BM
Markey Center for Molecular Genetics, Department of Microbiology and Molecular Genetics, University of Vermont, Burlington 05405.
Berzal-Herranz
A
A
Keller
C F
CF
Burke
J M
JM
D001482:Base Composition; D001483:Base Sequence; D002678:Chimera; D003854:Deoxyribonucleotides; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010766:Phosphorylation; D012313:RNA / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D012266:Ribose
D003854:Deoxyribonucleotides; D016337:RNA, Catalytic; D012313:RNA; D012266:Ribose; D008274:Magnesium