Binding of Transfer RNA to Carbon Films for Electron Microscopy: A Quantitative Approach

Abstract

It is essential to establish criteria for the identification of macromolecules studied by electron microscopy. The salt solutions, or (depending on the techniques used) the grain of the shadowing or the staining material or even dirt, may limit the level of confidence with which definitive statements about the macromolecules can be made. With this in mind, we have devised preparatory procedures for the study of one macromolecule, namely tRNA, and investigated its interaction with carbon films. (See also the accompanying paper, Yoshikami and Abermann, these Proceedings.)Highly purified monomeric mixed tRNA from yeast in a buffer of 0.1 M NaCl, 10 mM MgCl2, 10 mM Na cacodylate, pH 6.6 was used throughout this investigation. Freshly evaporated carbon films were stripped from mica and mounted on grids. tRNA was mounted by floating the grid on a droplet of the tRNA solution. Removal of excess salts and dehydration were done by successive rinses of the grid in ethanol. Ethanol was selected as a rinsing agent since the constituents of the buffer were all soluble in ethanol, and ethanol precipitation and subsequent drying is a well established procedure for concentrating tRNA without destroying its biological activity.