IUBMB Life, 48: 163–168, 1999 Copyright c ° 1999 IUBMB 1521-6543/99 $12.00 + .00 Original Research Article DNA-Dependent RNA Polymerase II from Candida Species Is a Multiple Zinc–Containing Metalloenzyme Meera Patturajan, Mayalagu Sevugan, and Dipankar Chatterji Center for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India Summary We have puried DNA-dependent RNA polymerase II from Candida albicans, a human pathogenic yeast. The enzyme consists of 9 polypeptides that are unique to C. albicans, their mobility on SDS-PAGE being different from the mobility of the corresponding subunits of RNA polymerase II from Saccharomyces cerevisiae or C. utilis. In the present study we also demonstrate that RNA pol II from C. albican and C. utilis are metalloproteins containing « 5 mol of zinc per mole of enzyme. Although prolonged dialysis in 10 or 20 mM EDTA failed to remove Zn(II) from the C. albicans enzyme, in the C. utilis enzyme 3 Zn(II) ions could be removed and then reconstituted in the presence of excess Zn(II). o-Phenanthroline (5 mM) removed Zn(II) from C. albicans enzyme irreversibly in a time-dependent fashion with concomitant loss of enzyme activity. Circular dichroism studies revealed structural changes on removal of zinc, thus suggesting a role for Zn in maintenance of structural stability. Further, we demonstrate that the largest subunit of the C. utilis enzyme and the 3 large subunits of the C. albicans enzyme can bind radioactive zinc. IUBMB Life, 48: 163–168, 1999 Keywords Candida albicans; Candida utilis; metalloenzyme; RNA polymerase II; zinc. INTRODUCTION Among several Candida species, Candida albicans is the most pathogenic. This opportunistic pathogen of human be- ings and other warm-blooded animals causes a spectrum of dis- eases, ranging from supercial infections of skin and mucosa to severe systemic infections (1). Though the pathogenic potential of C. albicans has made it a medically important organism, very little is known about the RNA polymerase II (EC 2.7.7.6; Received 4 January 1999; accepted 26 January 1999. Current address and address for correspondence: Meera Patturajan, Molecular Biology and Genetics, School of Medicine, Johns Hopkins University, 725 N. Wolfe St., Baltimore, Maryland 21205, U.S.A. Fax: (410)502-6718. E-mail: meera@welchlink.welch.jhu.edu pol II) 1 from this organism. This polymerase has been puried from various organisms, including animals and plant tissues, cultured cells, and lower eukaryotes (2–4); it is reported to con- tain zinc at a variable stoichiometry (5). The role of these zinc ions is still not well understood. In Escherichia coli metal sub- stitution experiments and spectroscopic studies suggested that the 2 Zn(II) atoms present are not functionally equivalent (6, 7 ). We have recently shown that yeast pol II has two tightly bound zinc ions (8). The potential zinc-binding domains of the largest and second largest pol II subunits in yeast have been analyzed in some detail. The largest subunit bears a consensus domain CX2 CX6¡ 12 CX-GHXGX24¡ 37 CX2 C in the amino-terminal re- gion, and the second largest subunit contains a motif of the form CX 2 CX 8¡ 25 CX 2 C in the carboxyl-terminal region (9, 10). Small protein domains encompassing these motifs on subunits B220 and B150 bind 65 Zn(II) in vitro when overproduced in E. coli (10). An amino acid substitution for each of the four cysteines of B150 leads to lethal or temperature-sensitive growth phenotypes and reduces zinc binding in vitro (9, 10). Also, temperature- sensitive mutations resulting from random mutagenesis are lo- cated at invariant positions within the metal-binding domain of the subunit B220 (11). These data suggest that at least for the two large subunits, the two potential metal-binding motifs are geni- une sites of zinc binding and, moreover, that they are essential for the proper functioning of the RNA polymerase. In the course of our work with nonsporulating yeast, C. albi- cans and C. utilis, we observed the presence of a large number of Zn(II) associated with pol II. Hence the present study is mo- tivated by an attempt to estimate exact number of Zn(II) present per molecule of enzyme and assign a function to these ions, if any. MATERIALS AND METHODS Chemicals. Tris, EDTA, acrylamide, sodium dodecyl sul- fate (SDS), and polyethylenimine were purchased from Sigma 1 Abbreviations: PAGE, polyacrylamide gel electrophoresis; pol II, RNA polymerase II; SDS, sodium dodecyl sulfate 163