Interaction of tRNAs and of phosphorothioate-substituted nucleic acids with an organomercurial. Probing the chemical environment of thiolated residues by affinity electrophoresis

Abstract

The interactions of 4-thiouridine and 5-[(methylamino)methyl]-2-thiouridine in tRNA and of phosphorothioate esters in nucleic acids with an organomercurial have been investigated. For this purpose, an affinity electrophoretic system has been developed in which the mercury derivative has been covalently immobilized in a standard polyacrylamide gel. The retardation of thiolated macromolecules was found to be sensitive to the chemical environment of the sulfur atom, giving characteristic interaction constants dependent on the nature of the modification and its accessibility to binding. The interaction could, in the case of tRNA, be abolished by conventional specific chemical modification of the thiolated bases, as well as by irradiation with 32P-derived beta-emission. Not only has the fractionation of sulfur-modified from unmodified species been attained but a quantitative application of the technique has made it possible to study the binding of mercury and, by competition, that of magnesium in terms of the conformation of tRNA.