Inactivation of Tetrahymena rRNA self-splicing by cis-platin proceeds through dissociable complexes.
The anti-cancer drug cis-diamminedichloroplatinum (II) (cis-DDP) reacted with Tetrahymena self-splicing rRNA ribozyme, causing loss of self-splicing activity and formation of a number of platinated RNA species. The formation of one distinct platinated product, migrating at an apparent size of 2400 nt, was closely associated with ribozyme inactivation. This platinated RNA was resistant to T1 ribonuclease digestion, suggesting the presence of inter-strand Pt cross-links. The reaction rate of cis-DDP with the ribozyme followed first order kinetics and showed a saturation effect with increasing cis-DDP concentration, characteristic of an affinity-label type of interaction rather than bimolecular collision. The apparent KI for binding of cis-DDP to the ribozyme was 62 microM. Ribozyme treated with urea was not inactivated by cis-DDP, indicating that the native structure of the RNA is required for reaction with cis-DDP. Mg++, which binds to the ribozyme and causes conformational changes in the molecule, protected the ribozyme from inactivation by cis-DDP and also prevented the formation of platinated RNA. These results suggest that binding of cis-DDP to sites formed by certain secondary or tertiary structural elements of the RNA enhance the rate and the specificity of reaction of the reagent with the ribozyme.
Danenberg
P V
PV
Department of Biochemistry, University of Southern California School of Medicine, Los Angeles 90033.
Shea
L C
LC
Danenberg
K D
KD
Horikoshi
T
T
D000818:Animals; D002945:Cisplatin / Q000378:metabolism / Q000494:pharmacology*; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D010957:Plasmids; D012322:RNA Precursors / Q000096:biosynthesis; D012326:RNA Splicing; D016337:RNA, Catalytic / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D013768:Tetrahymena / Q000235:genetics*; D013890:Thiourea / Q000737:chemistry
D003432:Cross-Linking Reagents; D012322:RNA Precursors; D016337:RNA, Catalytic; D013890:Thiourea; D002945:Cisplatin