Use of polylysine for adsorption of nuclei acids and enzymes to electron microscope specimen films.

Abstract

Enzymes and nucleic acids, free and as bound in binary complexes, adsorb to EM specimen films in well-distributed fashion if a dilute solution of polylysine is previously applied to the films. Electron micrographs are exhibited that demonstrate the usefulness of the technique in visualizing double- and single-stranded DNA, Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) in negative stain and polymerase complexed to poly(dA-dT) and to an 1100 base-pair restriction fragment of bacteriophage T7 DNA containing the early promoters. The base-pair spacing of DNA prepared for EM by the polylysine method apparently was 0.326 nm. Promoter sites (4) on the T7 fragment were located at 215, 440, 560 and 670 base-pair distances from the left terminus. When poly(dA-dT) was incubated with a 20:1 weight ratio of polymerase the bound enzyme particles were found to be about 2/3 as closely packed as is sterically permissible.