Capturing hammerhead ribozyme structures in action by modulating general base catalysis.
We have obtained precatalytic (enzyme-substrate complex) and postcatalytic (enzyme-product complex) crystal structures of an active full-length hammerhead RNA that cleaves in the crystal. Using the natural satellite tobacco ringspot virus hammerhead RNA sequence, the self-cleavage reaction was modulated by substituting the general base of the ribozyme, G12, with A12, a purine variant with a much lower pKa that does not significantly perturb the ribozyme's atomic structure. The active, but slowly cleaving, ribozyme thus permitted isolation of enzyme-substrate and enzyme-product complexes without modifying the nucleophile or leaving group of the cleavage reaction, nor any other aspect of the substrate. The predissociation enzyme-product complex structure reveals RNA and metal ion interactions potentially relevant to transition-state stabilization that are absent in precatalytic structures.
Department of Molecular and Cellular Biochemistry, Center for Structural Biology, University of Kentucky, Lexington, Kentucky, United States of America. ychi@uky.edu
Young-In
YI
Chi
Monika
M
Martick
Monica
M
Lares
Rosalind
R
Kim
William G
WG
Scott
Sung-Hou
SH
Kim
D001483:Base Sequence; D002384:Catalysis; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D015394:Molecular Structure; D017860:Nepovirus / Q000201:enzymology / Q000235:genetics; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D016337:RNA, Catalytic; C427204:hammerhead ribozyme