A stereochemical glimpse of the active site of the 8-17 deoxyribozyme from iodine-mediated cross-links formed with the substrate's scissile site.
A powerful approach for defining the active site of a complexly folded ribozyme or deoxyribozyme (DNAzyme) is to map the contact cross-links formed between the substrate's reaction site and component residues of the enzyme. Here, we use a novel iodine- and phosphorothioate-mediated method for generating contact cross-links to define key residues of the 8-17 DNAzyme most proximal to the scissile phosphodiester of its bound substrate. Substitution of a phosphorothioate for the scissile phosphodiester renders that site chiral. The cross-linking maps we obtain using chirally resolved substrates give us, for the first time, a stereochemical glimpse of the 8-17's active site. Thus, we identifiy the asymmetric positioning of the DNAzyme's C13 residue, which is catalytically indispensable. We also identify, for the first time, the previously unheralded C3 residue. On the basis of the latter's proximal location to the cleavage site and the impact of its mutation on the DNAzyme's catalytic rate, we hypothesize it may play an acid-base role in the catalysis of the 8-17 DNAzyme. Overall, the approach described in this paper should find wide application in the study of the tertiary folding of RNAs and DNAs, as well as of complexes formed by RNA and DNA with proteins and other ligands.
Sekhon
Gurpreet S
GS
Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.
Sen
Dipankar
D
D020134:Catalytic Domain / Q000235:genetics; D003432:Cross-Linking Reagents; D021881:DNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D013237:Stereoisomerism; D013379:Substrate Specificity
D003432:Cross-Linking Reagents; D021881:DNA, Catalytic; C407482:RNA-cleaving DNA 8-17