Studies on the effect of zinc ions on tRNA structure

The effects of divalent metal ions, particularly Zn²⁺ and Cd²⁺, on the structure of transfer rlbonucleic acid (tRNA) in solution, were investigated using: chromatographic retention of tRNA on reverse-phase 5 (RPC-5) columns, ultraviolet difference spectroscopy, fluorescence intensity of the Wye (Y) base in yeast phenylalanine tRNA (tRNA^Phe) thermal denaturation, and iminoproton nuclear magnetic resonance (¹H-NMR) spectroscopy as probes.

Increased chromatographic retention of several tRNAs on RPC-5 was caused by Zn²⁺ but not by either Cd²⁺ or Mg²⁺. Gradient and isocratic elution conditions were investigated. The chromatographic results were interpreted in terms of a model that evaluates the relative contributions of ionic and hydrophobic inter actions. Of the two kinds, the hydrophobic forces increased markedly and the ionic to a less extent. Of four tRNAs examined, tRNA^Phe (yeast) was unique in that the increase in retention was related biphasically to Zn²⁺ concentration whereas the other tRNAs showed a monophasic dependence. Both Cd²⁺ and Mg²⁺ eliminated the retention caused by 10 mM Zn²⁺ at 10 mM and 35 mM respectively. The biphasic nature of this reversal by Cd ²⁺ and Mg²⁺ may indicate that the chromatographic effect is due to the binding of Zn²⁺ to two sites.

The Zn²⁺ binding sites in tRNA were also examined with the imino-proton spectra of yeast tRNA^Phe and E. coli tRNA^Val. The largest effects of Zn²⁺ were on the imino-proton resonances of (G-15)•(C-58), (G-19)•(C-56), (G-10)•(0-25), and (U-12)•(A-23). Zn²⁺ appears to bind at both the Mg²⁺ sites in the loop formed by residues A-9, G-10, A-11, and U-12 (P-10 loop) and to the Mg²⁺ site in the "elbow" of the L structure near G-19, and G-20. Zn²⁺ may also bind in the terminal region of the extended D-helix, near G-15.

The increases in absorbance of tRNA at 294 nm, due to Zn²⁺, Cd²⁺ and Ni²⁺, suggest that these metals bind to sites on the bases, most probably the N-7 position of guanine; Mg²⁺ does not bind at this site. Since the increased absorbance of the 294-nm band saturates at micromolar metal ion concentrations these interactions are not responsible for the chromatographic effect that is not completed even at 20 mM.