Studies on the effect of zinc ions on tRNA structure

Author

John Michael Flanagan

Date of Award

3-1986

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

K. Bruce Jacobson

Committee Members

Larry Waters, Salil Niyogi, Gerard Bunick, Don Olins

Abstract

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.

Recommended Citation

Flanagan, John Michael, "Studies on the effect of zinc ions on tRNA structure. " PhD diss., University of Tennessee, 1986.
https://trace.tennessee.edu/utk_graddiss/12245