Pb(II)-catalysed cleavage of the sugar–phosphate backbone of yeast tRNAPhe—implications for lead toxicity and self-splicing RNA

Abstract

Pb(II) is extremely efficient at depolymerizing RNA and studies on tRNAs have shown that site-specific cleavages in these molecules can be brought about by the action of Pb(II). We have observed, by difference Fourier analysis, sugar-phosphate strand scission between residues 17 and 18 in crystals of yeast tRNAPhe soaked in dilute Pb(II) solution at pH 7.4. We have also deduced the structure of the Pb(II)-tRNAPhe derivative at pH 5.0 where this cleavage reaction is considerably slower and report that, in this structure, the sugar-phosphate backbone remains intact. We have, therefore, a picture of the reactants (at pH 5.0) and products (at pH 7.4) of this cleavage reaction. From this crystallographic study, and associated biochemical work, we have formulated a possible mechanism for the cleavage reaction and also present here some general ideas on the action of metal ions on nucleic acids.

Identification of the magnesium, europium and lead binding sites in E. coli and lupine tRNAPhe by specific metal ion‐induced cleavages

“…The Pb ion at the Pb2-binding site in yeast tRNA Phe has the shortest contacts to two bases of the variable region, A44 and G45, being directly coordinated to G45 06 [8][9][10]. The absence of any Pb-induced cleavages in that region in yeast tRNA Phe [14][15][16] was confirmed in the present study.…”

“…The lupine tRNA vh" having the same nucleotide sequence as yeast tRNA eh" in both the T-loop and Dloop, i.e. at the Pb-ion-binding site and at the cleavage site [8][9][10][11], is cleaved with the same strength and specificity. On the other hand, E. coil tRNA Ph~ is significantly more resistant to the reaction with Pb.…”

“…The Pb cleavages in the D-loop are induced in yeast tRNA Phe by a Pb ion tightly bound to the two T-loop bases U59 and C60 [8][9][10]. In E. coli tRNA Phi, the latter base is replaced by U60, and most likely this single base substitution is sufficient to change the position and binding constant of the tightly bound Pb ion in a way that results in a much lower cleavage specificity and efficiency.…”

“…On the other hand, in E. coli tRNA Phe where G45 is replaced by uridine and A44 by guanosine, a series of weak Pb, Eu and Mg-induced cleavages occur in that region. It is, therefore, tempting to assume that due to the above base substitutions a metal-ion-binding site is created in E. coli tRNA Phe that is highly similar for Pb, Eu and Mg ions but different from the Pb2-(Sm4)-binding site in yeast tRNA Phe [8][9][10]18,19].…”

“…The metal hydrates bind to suitable pockets formed by tRNA tertiary structure [3,4] and several metal ions, especially Pb, Mg and Eu are capable of cleaving the tRNA chain with a high degree of specificity [5][6][7]. The mechanism of the specific yeast tRNA ph¢ hydrolysis by Pb was proposed some years ago [8][9][10][11]. It involves initial proton abstraction from a sugar 2'-OH group at the cleavage site by the ionized Pb hydrate, acting from its tight binding site.…”