Effect of zinc ions on tRNA structure : I. Reversed-phase chromatography

Abstract

The effect of zinc on the chromatographic behavior of four tRNAs was examined on RPC-5 and Aminex A-28 columns. RPC-5 contains dichlorodifluoroethylene beads coated with a quaternary ammonium compound where the substituents are: R 1 = methyl, and R 2-4& z.dbnd; C 8-10 hydrocarbons. Aminex A-28 contains quaternary ammonium covalently attached to styrene-divinylbenzene copolymer lattice and R 1-3 are methyl groups. The retentions of tRNA Val, tRNA Ile, and tRNA Lys of E. coli and yeast tRNA Phe on RPC-5 were all markedly increased by Zn 2+ ions. In contrast, no increased retention due to Zn 2+ was observed when tRNA Phe was chromatographed on Aminex A-28. A model for chromatography on RPC-5 is developed which treats the elution behavior of tRNAs from this matrix as the sum of ionexchange and hydrophobic interactions. The chromatography of tRNA in the presence and absence of Zn 2+ is interpreted in terms of this model and the effects of sodium chloride concentration, temperature, and pH were explored as the experimental variables. These experiments suggest that in the absence of Zn 2+ tRNA does not interact appreciably with the hydrophobic surface of the column. The addition of Zn 2+ has three effects on chromatography: (1) a decrease in the number of anionic sites on the tRNA which interact with the positively charged ammonium ion, (2) an increase in affinity of the tRNA for these ionic sites, and (3) an increase in affinity of tRNA for hydrophobic sites on the column. All three effects were fully reversed by the addition of Cd 2+ (10 m M) or Mg 2+ (35 m M), but only partially reversed at lower concentrations of these competing ions. These results show that chromatography on RCP-5 can be a sensitive physical chemical technique for examination of the structure of tRNA, and probably for other nucleic acids as well.