Crystallographic and biochemical investigation of the lead(II)-catalyzed hydrolysis of yeast phenylalanine tRNA

Abstract

X-ray diffraction data from monoclinic crystals of yeast tRNAPhe soaked in dilute lead(II) acetate solutions at pH 5.0 and at pH 7.4 have been collected to a resolution of 3 A, and the Pb(II) binding sites have been obtained by difference Fourier analyses. The same three Pb(II) binding sites are observed at both of these pH values. At pH 7.4 an extra peak of negative electron density appears on the difference map close to one of the Pb(II) binding sites and at the position of phosphate-18, indicating cleavage of the sugar-phosphate-chain between residues D-17 and G-18 of the tRNAPhe molecule in this derivative. Chain scission does not occur to any observable extent in the structure at pH 5.0, and we have, therefore, a picture of the reactants (at pH 5.0) and products (at pH 7.4) of this cleavage reaction. Polyacrylamide gel electrophoresis as well as sequencing experiments confirms the cleavage of the tRNAPhe molecule into one-fourth and three-fourth fragments, with the shorter fragment consisting essentially of residues G-1 through D-17 while the larger fragment contains residues G-18 through A-76. End-group analyses suggest a ribose cyclic 2',3'-phosphate at D-17 of the one-fourth fragment with a 5'-OH at G-18 of the three-fourth fragment. Cleavage of the tRNAPhe molecule does not occur in the absence of Pb(II), and the proximity of one of these metal ions to the cleavage site strongly implicates this metal ion in the cleavage reaction. Consideration of several possible mechanisms for the reaction, taking into account the biochemical and crystallographic facts presented above, suggests that the cleavage involves removal of the proton from the 2'-OH of ribose-17 by a Pb(II)-bound hydroxyl group. Subsequent nucleophilic attack of the resulting 2'-O- on the phosphorus atom of phosphate-18, presumably through a pentacoordinate phosphorus cyclic intermediate (as in the action of pancreatic ribonuclease A), results in chain scission. It cannot be decided whether the displacement, within the pentacoordinate intermediate, proceeds via an in-line or adjacent pathway, but an exploration of the likelihood of either pathway is presented. Strand cleavage at the particular site occurs fortuitously because the aquo Pb(II) ion binds at the correct distance and presumably in such a manner as to present a hydroxyl group in the correct orientation to effect the proton abstraction.(ABSTRACT TRUNCATED AT 400 WORDS)

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

“…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.…”

“…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.…”

An extended structural signature for the tRNA anticodon loop

“…Secondary structure of tRNA anticodon hairpins for which coordinates are deposited in the Nucleic Acid Database (NDB) (Berman et al+, 1992) Brown et al+, 1985;trna04: Sussman et al+, 1978;trna05: Comarmond et al+, 1986;trna06: Westhof & Sundaralingam, 1986;trna07, trna08, trna09: Westhof et al+, 1988;trna10: Hingerty et al+, 1978;pr0004: Nissen et al+, 1999;pr0024: Goldgur et al+, 1997;ptr012: Nissen et al+, 1995+ b In tRNAs, purines represent roughly a little more than 50% of the nucleotides present at position 35 (Auffinger & Westhof, 1998b) as expected from the genetic code and deduced from the 550 tRNAs and 3,704 tDNA gene sequences itemized in the tRNA database (Sprinzl et al+, 1998)+ From the nine available crystallographic structures of yeast tRNA Phe (including the structures extracted from the yeast tRNA Phe /EF-TU and the Thermus thermophilus tRNA Phe /RS complexes where the anticodon loop structure is not altered, ptr012 and pr0024, respectively, see Table 1), it has been inferred that the 29-hydroxyl group of U33 forms a hydrogen bond with the N7 atom of A35 (Fig+ 2)+ This assumes that the hydroxyl (U33)O29-H group points toward the (A35)N7…”

Site‐specific cleavage reaction catalyzed by leadzyme is enhanced by combined effect of lead and rare earth ions

“…Previously, the binding of metal ions to RNA was ~ 30. studied for tRNA and it was reported that tRNA had many ~ 20. different binding sites for metal ions [17]. For example, in the ~o-presence of both Pb 2+ and Mg 2+, Pb 2+ coordinated directly to 0 4 of U and N 3 of C in the T loop at the corner of the L-0-structure of tRNA, while Mg 2+ located near the site of Pb 2+ [18]. The two classes of metal ion binding sites were also observed in Tetrahymena self-splicing intron [6,7,19].…”