Open AccessProbing the mechanism of an Mn 2+ ‐dependent ribozyme by means of platinum complexes
Author(s) -
Bombard Sophie,
Kozelka Jiří,
Favre Alain,
Chottard JeanClaude
Publication year - 1998
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1998.2520025.x
Subject(s) - ribozyme , guanosine , chemistry , purine metabolism , stereochemistry , cleavage (geology) , platinum , residue (chemistry) , crystallography , catalysis , medicinal chemistry , enzyme , rna , biochemistry , biology , gene , paleontology , fracture (geology)
The smallest ribozyme system known is the pentanucleotide GAAACp, which is specifically cleaved by Mn 2+ , 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 Mn 2+ 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 Mn 2+ by Pt 2+ . We thus replaced the proposed kinetically labile G1N7‐Mn 2+ ‐A4N7 cross‐link by an inert N7‐ trans ‐Pt(NH 3 ).es2+.rb.ei2.rb‐N7 cross‐link. In a complementary investigation, the N7 atoms of the individual purines of GAAACp were selectively blocked by a Pt(NH 3 ).es2+.rb.ei3.rb residue to determine which of the N7 sites participate in the Mn 2+ ‐mediated cleavage. Other N7‐Pt II ‐N7 crosslinks were also investigated. Accordingly, we have prepared four monoadducts, each bearing the Pt(NH 3 ).es2+.rb.ei3.rb residue on one of the purines and a series of chelates of trans ‐Pt(NH 3 ).es2+.rb.ei2.rb and cis ‐Pt(NH 3 ).es2+.rb.ei2.rb and have tested them for Mn 2+ ‐induced cleavage. Binding of Pt(NH 3 ).es2+.rb.ei3.rb to G1 or A4 did not alter the efficiency of the specific cleavage between G1 and A2 catalyzed by Mn 2+ /poly(U), whereas cross‐linking of G1 and A4 by trans ‐Pt(NH 3 ).es2+.rb.ei2.rb inhibited it completely. Hence, a cross‐link between G1 and A4 is not required for the site‐specific cleavage. Binding of Pt(NH 3 ).es2+.rb.ei3.rb 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 Mn 2+ ‐dependent cleavage between A4 and C5 was observed for the G1‐A4 and G1‐A3 adducts cross‐linked by trans ‐Pt(NH 3 ).es2+.rb.ei2.rb, the G1‐A2 adduct cross‐linked by cis ‐Pt(NH 3 ).es2+.rb.ei2.rb, and the three monoadducts bearing the Pt(NH 3 ).es2+.rb.ei3.rb residue on G1, A2 or A3; poly(U) did not exert any influence on this cleavage.