RAT LIVER RNA POLYMERASES zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Inactivation of Rat Liver zyxwv RNA Polymerases I and I1 and Yeast RNA Polymerase I by Pyridoxal 5’-Phosphate. Evidence for the Participation of Lysyl Residues at the Active Site? Joseph Martial,* Josefina Zaldivar, Paulina Bull, Alejandro Venegas, and Pablo Valenzuela* ABSTRACT: Purified DNA-dependent RNA polymerase forms I (A) and I1 (B) from rat liver and form I from yeast are rapidly inactivated by pyridoxal 5’-phosphate at pH 8.0. The inhibition is relatively specific since pyridoxamine zyxwvuts 5’- phosphate is not an inhibitor and pyridoxal is about 12 times less effective than pyridoxal 5’-phosphate. The inacti- vation is reversed by high concentrations of amines, and can be made irreversible by reduction with NaBH4. Spectral analysis of the inhibited enzyme and its NaBH4 reduction product indicates that a Schiff base forms between the alde- hyde group of pyridoxal 5‘-phosphate and one or more amino groups of the protein. N€-Pyridoxyllysine was identi- fied as the only product in acid hydrolysates of the reduced K n o w l e d g e about the amino acid composition and topog- raphy of active sites is important for understanding the na- ture of enzyme catalysis and substrate specificity. In recent years, a good deal of work has been carried out on the puri- fication of animal RNA polymerases, especially those from rat liver and calf thymus (Roeder and Rutter, 1969, 1970; Kedinger et al., 1972; Gissinger and Chambon, 1972). Al- though the study of many of their properties and subunit structure is well-advanced (Chesterton and Butterworth, 1971; Weaver et al., 1971; Kedinger and Chambon, 1972; Schwartz and Roeder, 1974, 1975), little is known about their active sites and mechanism of action. Recently, Valen- zuela et al. (1973) provided evidence for the presence of a metal ion at the active site of animal RNA polymerases, probably zinc by analogy with the Escherichia coli enzyme. Searching for other groups at the active center of these enzymes, we have selected pyridoxal 5’-phosphate as a mod- ifying agent. We have previously reported that this com- pound is a specific inhibitor of E. coli RNA polymerase, presumably by reacting with a critical amino group at the active center of the enzyme (Venegas et al., 1973; Bull et a]., 1975). We now present results showing that pyridoxal 5’-phosphate is also a specific inhibitor of rat liver RNA po- lymerases I (A) and I1 (B) and yeast RNA polymerase I (A). The evidence presented suggests that pyridoxal zyxwvut 5’- phosphate inhibition results from the modification of one or more unique lysyl amino groups presumably located at the active site of the enzymes. A preliminary account of this work has been reported (Valenzuela et al., 1974). From the Laboratorio de Bioquimica, Departamento de Biologia Celular. Universidad Catolica, Casilla I 14-D. Santiago, Chile. Re- ceived June 23. 1975. This research was financed by Fondo de Investi- gaciones Universidad Catolica, CONICYT Chile, and the Cell Biology Program of O.A.S. in Chile. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA * Visiting scholar under a Technical Assistance Program between the governments of Belgium and Chile (1 97 I - 1973). yeast RNA polymerase I-pyridoxal 5’-phosphate complex. Complete inactivation of yeast polymerase I results in the incorporation of 3-4 mol of pyridoxal 5’-phosphate/ 1 mol of enzyme. DNA and nucleotide substrates partially protect the enzymes from inactivation. These results suggest that one or more lysyl amino groups are critical for the activity of animal RNA polymerases and show that pyridoxal 5’- phosphate is a suitable probe for studying the active sites of these enzymes. Comparison.of the present results with those previously obtained with Escherichia coli RNA polymerase in this laboratory suggest a new degree of structural homol- ogy between eucaryotic and procaryotic RNA polymerases. Materials and Methods Materials. [3H]UTP (20 Ci/mmol) and NaB3H4 (185 and 250 mCi/mmol) were obtained from New England Nu- clear Corp., Boston, Mass. Nucleoside triphosphates, di- thiothreitol, calf thymus DNA, and pyridoxal-HCI were purchased from Sigma Chem. Co., St. Louis, Mo. Pyridoxal 5’-phosphate and pyridoxamine 5’-phosphate were generous gifts from Professors F. Marcus and N. Carvajal, respec- tively. DEAE-cellulose (DE-52) was a Whatman product, and DEAE-Sephadex A-25 was obtained from Pharmacia Fine Chemicals. Sucrose, glycerol, and ammonium sulfate were enzyme-grade reagents. Yeast cells were kindly pro- vided by Red Star Yeast Co., Oakland, Calif. Enzyme Purification. Yeast RNA polymerase I was pre- pared by a modification of the method of Buhler et al. (1974). The last two steps of this procedure are ion-filtra- tion chromatography on DEAE-Sephadex and sucrose gra- dient centrifugation instead of DEAE-cellulose chromatog- raphy and glycerol gradient centrifugation. The enzyme ob- tained is homogeneous in acrylamide gel eleetrophoresis and has a specific activity of 300 units/mg of protein. One unit of activity is defined as 1 nmol of UMP incorporated per 10 min at 30° into RNA. The details of the purification procedure will be reported elsewhere (P. Valenzuela and W. J. Rutter, manuscript in preparation). Rat liver RNA polymerases I and I1 were partially puri- fied from isolated nuclei by a method which combines pro- cedures of Roeder and Rutter (1969), Kedinger et al. (1972), Gissinger and Chambon (1972), and Kedinger and Chambon (1972). Enzyme I was purified by (NH4)2S04 fractionation, DEAE-cellulose chromatography, DEAE- Sephadex chromatography, and glycerol gradient centrifu- gation. Enzyme I1 was purified by (NH4)2S04 fraction- ation, DEAE-cellulose chromatography, and glycerol gradi- ent centrifugation. The various steps employed are de- scribed in detail in the following paragraphs. BIOCHEMISTRY, VOL. 14, NO. 22, 1975 4907