Functional involvement of G8 in the hairpin ribozyme cleavage mechanism.
The catalytic determinants for the cleavage and ligation reactions mediated by the hairpin ribozyme are integral to the polyribonucleotide chain. We describe experiments that place G8, a critical guanosine, at the active site, and point to an essential role in catalysis. Cross-linking and modeling show that formation of a catalytic complex is accompanied by a conformational change in which N1 and O6 of G8 become closely apposed to the scissile phosphodiester. UV cross-linking, hydroxyl-radical footprinting and native gel electrophoresis indicate that G8 variants inhibit the reaction at a step following domain association, and that the tertiary structure of the inactive complex is not measurably altered. Rate-pH profiles and fluorescence spectroscopy show that protonation at the N1 position of G8 is required for catalysis, and that modification of O6 can inhibit the reaction. Kinetic solvent isotope analysis suggests that two protons are transferred during the rate-limiting step, consistent with rate-limiting cleavage chemistry involving concerted deprotonation of the attacking 2'-OH and protonation of the 5'-O leaving group. We propose mechanistic models that are consistent with these data, including some that invoke a novel keto-enol tautomerization.
Pinard
R
R
Department of Microbiology and Molecular Genetics, The University of Vermont, 306 Stafford Hall, Burlington, VT 05405, USA.
Hampel
K J
KJ
Heckman
J E
JE
Lambert
D
D
Chan
P A
PA
Major
F
F
Burke
J M
JM
D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006151:Guanosine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010641:Phenotype; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011522:Protons; D012313:RNA / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry*; D012965:Sodium Chloride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D014466:Ultraviolet Rays; D014867:Water / Q000378:metabolism
D011522:Protons; D016337:RNA, Catalytic; C418481:hairpin ribozyme; D014867:Water; D006151:Guanosine; D012965:Sodium Chloride; D012313:RNA