Probing the mechanism of an Mn2+-dependent ribozyme by means of platinum complexes.
The smallest ribozyme system known is the pentanucleotide GAAACp, which is specifically cleaved by Mn2+, in the presence of poly(U), generating guanosine 2',3'-cyclic phosphate and AAACp. A plausible mechanism has been proposed, involving the participation of two Mn2+ with structural and catalytic roles, the first one cross-linking the two N7 atoms of G1 and A4, and the other binding to the N7 atom of A2 and activating the 2'-OH group of G1 [Kazakov, S. & Altman, S. (1992) Proc. Natl Acad. Sci. USA 89, 7939-7943]. In the present work, we have utilized the high affinity of Pt(II) complexes for N7 atoms of purines in an attempt to form a stable active ribozyme by replacing the structural Mn2+ by Pt2+. We thus replaced the proposed kinetically labile G1N7-Mn2+-A4N7 cross-link by an inert N7-trans-Pt(NH3)(2)(2+)-N7 cross-link. In a complementary investigation, the N7 atoms of the individual purines of GAAACp were selectively blocked by a Pt(NH3)(3)(2+) residue to determine which of the N7 sites participate in the Mn2+-mediated cleavage. Other N7-Pt(II)-N7 crosslinks were also investigated. Accordingly, we have prepared four monoadducts, each bearing the Pt(NH3)(3)(2+) residue on one of the purines and a series of chelates of trans-Pt(NH3)(2)(2+) and cis-Pt(NH3)(2)(2+) and have tested them for Mn2+-induced cleavage. Binding of Pt(NH3)(3)(2+) to G1 or A4 did not alter the efficiency of the specific cleavage between G1 and A2 catalyzed by Mn2+/poly(U), whereas cross-linking of G1 and A4 by trans-Pt(NH3)(2)(2+) inhibited it completely. Hence, a cross-link between G1 and A4 is not required for the site-specific cleavage. Binding of Pt(NH3)(3)(2+) to A2 or A3 strongly inhibits the G1/A2 cleavage, suggesting that these bases are likely to be involved in manganese coordination in the cleaving complex. A site-specific Mn2+-dependent cleavage between A4 and C5 was observed for the G1-A4 and G1-A3 adducts cross-linked by trans-Pt(NH3)(2)(2+), the G1-A2 adduct cross-linked by cis-Pt(NH3)(2)(2+), and the three monoadducts bearing the Pt(NH3)(3)(2+) residue on G1, A2 or A3; poly(U) did not exert any influence on this cleavage.
Bombard
S
S
Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques, Université René Descartes, URA 400 CNRS, Paris, France.
Kozelka
J
J
Favre
A
A
Chottard
J C
JC
D002384:Catalysis; D002945:Cisplatin / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology*; D008958:Models, Molecular; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009843:Oligoribonucleotides / Q000378:metabolism; D017671:Platinum Compounds / Q000378:metabolism*; D011072:Poly U / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D016337:RNA, Catalytic / Q000378:metabolism*; D014508:Urea / Q000494:pharmacology
D003432:Cross-Linking Reagents; D009843:Oligoribonucleotides; D017671:Platinum Compounds; D016337:RNA, Catalytic; C034697:transplatin; D011072:Poly U; D008345:Manganese; D012313:RNA; D014508:Urea; D002945:Cisplatin