Energetics of RNA cleavage: implications for the mechanism of action of ribozymes.
A new class of ribozymes produce 2',3'-cyclic phosphate upon self-catalyzed cleavage of RNA molecules, similar to those observed during enzymatic (RNase-catalyzed) as well as non-enzymatic hydrolyses of RNAs. This product suggests that the reaction intermediate/transition state is a pentacoordinated oxyphosphorane. In order to elucidate the energetics of these RNA cleaving reactions, the reaction coordinate has been simulated and a pentacoordinated intermediate has been characterized via ab initio molecular orbital calculations utilizing the dianionic hydrolysis-intermediate of methyl ethylene phosphate as a model compound. The calculated reaction coordinate indicates that the transition state for the P-O(2') bond cleavage is lower in energy than that for the P-O(5') bond cleavage under uncatalyzed conditions. Thus, the dianionic pentacoordinated phosphorus intermediate tends to revert back to the starting RNA by cleaving the P-O(2') bond rather than productively cleaving the P-O(5') bond. In order for ribozymes to effectively cleave RNA molecules, it is therefore mandatory to stabilize the leaving 5'-oxygen, e.g. by means of a divalent magnesium ion.
Taira
K
K
Fermentation Research Institute, Agency of Industrial Science and Technology, MITI, Tsukuba Science City, Fukuoka, Japan.
Uebayasi
M
M
Maeda
H
H
Furukawa
K
K
D002384:Catalysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D008274:Magnesium / Q000378:metabolism; D008968:Molecular Conformation; D015394:Molecular Structure; D010710:Phosphates / Q000378:metabolism; D012313:RNA / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism
D010710:Phosphates; D016337:RNA, Catalytic; D012313:RNA; D008274:Magnesium