Abstract
A rapid and simple, large-scale method for the purification of DNA-dependent RNA polymerase III (EC 2.7.7.6) from wheat germ is presented. The method involves enzyme extraction at low ionic strength, polyethyleneimine fractionation, (NH4)2SO4 precipitation and chromatography on DEAE-Sepharose CL-6B, DEAE-cellulose and heparin agarose. Milligram quantities of highly purified enzyme were obtained from kilogram quantities of starting material in 2-3 days. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that RNA polymerase III contains 14 subunits with MW of 150,000, 130,000, 94,000, 55,000, 38,000, 30,000, 28,000, 25,000, 24,500, 20,500, 20,000, 19,500, 17,800 and 17,000. Subunit structure comparison of wheat germ RNA polymerases I, II and III indicates that all 3 enzymes may contain common subunits with MW 20,000, 17,800 and 17,000. RNA polymerases II and III may contain a common subunit with a MW 25,000 and RNA polymerases I and III may contain a common subunit with a MW of 38,000.