WHEAT GERM RNA POLYMERASE zyxwvutsrq I1 PURIFICATION A New Method for the Large-Scale Purification of Wheat Germ DNA-Dependent RNA Polymerase II? Jerome J. Jendrisakt and Richard R. Burgess* ABSTRACT: An improved method for the purification of the a-amanitin-sensitive deoxyribonucleic acid dependent ribo- nucleic acid polymerase [ribonucleosidetriphosphate:RNA- nucleotidyltransferase, EC 2.7.7.61 (RNA polymerase I1 or RNA polymerase B) from wheat germ is presented. The method involves homogenization of wheat germ in a buffer of moderate ionic strength, precipitation of RNA polymer- D e t a i l e d studies of the chemical and physical properties of eukaryotic DNA-dependent RNA polymerases require that large amounts of enzyme be conveniently prepared in high purity, Eukaryotic nuclei contain multiple RNA po- lymerases with different functions. Thus RNA polymerase I, which is not inhibited by a-amanitin (Jacob et al., 1970), is localized in the nucleolus (Roeder and Rutter, 1970) and is responsible for the synthesis of ribosomal precursor RNA (Blatti et al., 1970). RNA polymerase 11, which is inhibited by low levels of a-amanitin (Jacob et al., 1970), is nucleo- plasmic (Roeder and Rutter, 1970) and synthesizes hetero- geneous nuclear RNA (Blatti et al., 1970). RNA polymer- ase 111, which is inhibited only by very high concentrations of a-amanitin, is believed to be responsible for synthesis of zyxwvuts 5 S and precursor for tRNA (Weinmann and Roeder, 1974). While RNA polymerases have been isolated and studied from a wide variety of eukaryotes (Chambon, 1974; Jacob, 1973), the low RNA polymerase content and limiting amounts and/or expense of the starting material prohibit large-scale purification of the RNA polymerases from many of these sources. Because RNA polymerase zyxwvut I1 is gen- erally present in larger amounts and is more stable to puri- fication than RNA polymerases I and 111, it has been the most extensively studied. From calf thymus tissue, which has been reported to be the richest source of RNA polymer- ases (Chambon, 1974), milligram quantities of pure RNA polymerase I1 have been obtained. However, most of the purification procedures involve many steps resulting in low yields of enzyme (Gissinger and Chambon, 1972; Weaver et al., 1971). Often the procedures cannot be scaled up due to sonication (Gissinger and Chambon, 1972; Weaver et al., 197 1) and preparative ultracentrifugation (Schmincke and Hausen, 1973) of large volumes. Wheat germ is a convenient source for the purification of RNA polymerase. The material is inexpensive, easily stored, available in virtually unlimited quantities, ready for use with no preliminary tissue isolation, and is rich in the t From the McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706. zyxwvutsrqpo Receiued June zyxwvutsrqp 18, zyxwvutsr 1975. This work was supported by National Institutes of Health Postdoctoral Fel- lowship Grant No. l F22 CA02804-01 and by National Institutes of Health Program Project Grant No. CA-07175. * National Institutes of Health Postdoctoral Fellow. ase with Polymin P (a polyethylenimine), elution of RNA polymerase from the Polymin P precipitate, ammonium sul- fate precipitation, and chromatography on DEAE-cellulose and phosphocellulose. RNA polymerase I1 is purified over 4000-fold with a 60% recovery, resulting in a yield of 25-30 mg of RNA polymerase from 1 kg of starting material. a-amanitin-sensitive RNA polymerase (Jendrisak and Becker, 1973). Although a method for the purification of this enzyme has been presented earlier (Jendrisak and Becker, 1974), the method described here is more efficient, results in higher yields, and is more suitable for scale up. The new method is designed to avoid sonication, ultra- centrifugation, and dialysis of large volumes. Fractionation of the crude extract with Polymin P (a method first intro- duced by Zillig et al. (1970) for the purification of Esche- richia coli RNA polymerase) results in a substantial purifi- cation with high yields and complete removal of nucleic acids. The high purification achieved at the initial stages of this procedure allows more efficient use of subsequent col- umn chromatographic steps, resulting in the complete puri- fication of RNA polymerase from several kilograms of starting material in 2 days. Since the enzymatic and physical properties of wheat germ RNA polymerase I1 have been shown to be similar, if not identical, to the analogous enzymes isolated from other eukaryotic sources (Jendrisak and Becker, 1973, 1974), any new information obtained from further studies of wheat germ RNA polymerase should be applicable to these other eukaryotic RNA polymerases. The availability of RNA polymerase in the large quantities obtained here should allow further studies and detailed analysis of the chemical, physical, and subunit properties of this enzyme. Experimental Procedures Materials. Raw wheat germ was obtained from VioBin Corporation, Monticello, Ill., and was stored in a cold room (4OC). Polymin P was kindly donated by Badische Anilin and Soda Fabrik, WHOZ Hauptlaboratorium B9, Hwh- schullieferungen, 6700 Ludwigshafen/Rhein, Germany, and was stored at room temperature. Ribonucleoside tri- phosphates (ATP, GTP, CTP, and UTP) were purchased from P-L Biochemicals and [5-3H]UTP (26.9 Ci/mmol) was purchased from New England Nuclear. Calf thymus DNA was purchased from Worthington, bovine serum al- bumin from Miles (Pentex) and a-amanitin from Henley and Company, New York. Tris base and EDTA were pur- chased from Sigma, dithiothreitol and Miracloth from Cal- biochem, (NH4)2S04 (enzyme grade), Brij 35, and Coom- assie Brilliant Blue R-250 from Schwartz/Mann, and eth- ylene glycol (99+%) was from Aldrich. DEAE-cellulose BIOCHEMISTRY, VOL. 14, NO. 21, 1975 4639