Activity of the hammerhead ribozyme upon inversion of the stereocenters for the guanosine 2'-hydroxyls.
Two guanosine 2'-hydroxyls in the hammerhead RNA complex at positions G5 and G8 are critical for efficient cleavage by this RNA catalyst. These two functional groups are likely involved in the binding of the metal cofactor, or they are involved in specific interresidue hydrogen-bonding interactions. The importance of the stereochemical positioning of both critical 2'-hydroxyls was investigated by comparing the cleavage rates of three arabinosylguanine-substituted complexes (in which the positions of specific guanosine 2'-hydroxyls were stereochemically altered by inverting the C2' stereocenter) with that of the native complex, as well as with the rates of the dG- and dFG-substituted complexes [in which the 2'-hydroxyls are absent as the result of substitution by 2'-deoxyguanosine (dG) or 2'-deoxy-2'-fluoroguanosine (dFG)]. The G5araG and G8araG complexes exhibit dramatically different cleavage rates. The G5araG complex is essentially inactive, at least 10(5)-fold slower than the native complex. RNA cleavage by this analogue ribozyme is also 1000-fold slower than cleavage by either the G5dG or the G5dFG ribozyme, both of which lack the 2'-hydroxyl at G5. By comparison, catlytic efficiency of the G8araG complex as expressed by kcat/Km is comparable with that of the native complex and some 2 orders of magnitude more active than either the G8dG or the G8dFG complex.
Fu
D J
DJ
Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02167.
Rajur
S B
SB
McLaughlin
L W
LW
D001089:Arabinose / Q000378:metabolism; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism*; D017665:Hydroxyl Radical / Q000378:metabolism; D012313:RNA / Q000138:chemical synthesis / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
D016337:RNA, Catalytic; D017665:Hydroxyl Radical; D012313:RNA; C083670:2'-deoxy-2'-fluoroguanosine; D001089:Arabinose; D003849:Deoxyguanosine