Temperature-gradient gel electrophoresis. Thermodynamic analysis of nucleic acids and proteins in purified form and in cellular extracts

Abstract

A temperature-gradient gel electrophoresis technique and its application to the study of structural transitions of nucleic acids and protein-nucleic acid complexes are described. The temperature gradient is established in a slab gel by means of a simple ancillary device for a commercial horizontal gel apparatus. The gradient may be freely selected between 10 and 80 degrees C, and is highly reproducible and linear. In a normal application the biopolymers migrate perpendicular to the temperature gradient so that every individual molecule is at constant temperature throughout electrophoresis. The structural transition of a biopolymer is seen as a continuous band which is retarded or speeded up in the temperature range of the transition. Dissociation processes are mostly irreversible under the conditions of electrophoresis and, therefore, show up as discontinuous transitions from a slow-moving to fast-moving band. As examples the conformational transitions of viroids, double-stranded RNA from reovirus, double-stranded satellite RNA from cucumber mosaic virus and repressor-operator complexes have been studied. It could be shown that by this method dsRNA molecules may be differentiated which differ only in one base-pair, or proteins differing in one amino acid only. As a particular advantage, temperature-gradient gel electrophoresis allows the study of conformational transitions of biopolymers which have not been purified. The biopolymer may either be identified by silver staining as a specific band among many others or, if the study is carried out on nucleic acids, these may be recorded by hybridization with a radioactive probe.