A conformational change in the "loop E-like" motif of the hairpin ribozyme is coincidental with domain docking and is essential for catalysis.
The catalysis of site-specific RNA cleavage and ligation by the hairpin ribozyme requires the formation of a tertiary interaction between two independently folded internal loop domains, A and B. Within the B domain, a tertiary structure has been identified, known as the loop E motif, that has been observed in many naturally occurring RNAs. One characteristic of this motif is a partial cross-strand stack of a G residue on a U residue. In a few cases, including loop B of the hairpin ribozyme, this unusual arrangement gives rise to photoreactivity. In the hairpin, G21 and U42 can be UV cross-linked. Here we show that docking of the two domains correlates very strongly with a loss of UV reactivity of these bases. The rate of the loss of photoreactivity during folding is in close agreement with the kinetics of interdomain docking as determined by hydroxyl-radical footprinting and fluorescence resonance energy transfer (FRET). Fixing the structure of the complex in the cross-linked form results in an inability of the two domains to dock and catalyze the cleavage reaction, suggesting that the conformational change is essential for catalysis.
Hampel
K J
KJ
Department of Microbiology and Molecular Genetics, University of Vermont, Burlington 05405, USA.
Burke
J M
JM
D002384:Catalysis; D020134:Catalytic Domain; D006868:Hydrolysis; D007700:Kinetics; D008027:Light; D009690:Nucleic Acid Conformation; D010777:Photochemistry; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism; D014466:Ultraviolet Rays
D016337:RNA, Catalytic; C418481:hairpin ribozyme