GENE ELSEVIER Gene 190 (1997) 77-85 The presence of two tightly bound Zn2+ ions is essential for the structural and functional integrity of yeast RNA polymerase II1 Sevugan Mayalagu, Meera Patturajan, Dipankar Chatterji * Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007 (A. P.), India Received 3 March 1996; received in revised form 18 June 1996; accepted 9 July 1996; Received by S.E. Hasnain Abstract DNA-dependent RNA polymerases (RNApol) are Zn’ + metalloproteins where the Zn2’ ion plays both catalytic and structural roles. Although the ubiquitous presence of Zn2+ with the RNApol from eukaryotes had already been established, the exact stoichiometry of Zn ” ion(s) per mole enzyme is not well documented, and its role in enzymatic function remains elusive. We show here that RNApolII from Saccharomyces cerevisiae has two Zn 2f ions tightly associated with it which are necessary for its transcriptional activity. Upon prolonged dialysis against 10 mM EDTA for 4-5 h, the enzyme loses one Zn’+ , as well as partial activity. However, Zn2+ can be added back to the enzyme, but without recovering its total activity. 5 mM orthophenanthroline (OP) removes one Zn2+ within 2 h; the enzyme, however, cannot be reconstituted back with Zn2+. Circular dichroism (CD) studies showed that the conformation of the native enzyme is unique and cannot be reproduced with ZnZC-reconstituted RNApolII. Similarly, the rate of abortive synthesis of a dinucleotide product over a non-specific template is faster when catalyzed by two Zn2 +-native enzymes. Zn2 +-reconstituted RNApolII or one Zn2+ -RNApolII showed a slower abortive synthesis rate. 65Zn2+-blotting experiments indicated that the removal of one Zn ‘+ from the enzyme destroys the Zn2+-binding ability of the larger subunits of yeast RNApolII. In order to check whether the presence of Zn 2+ ions has any effect on substrate recognition, we followed the binding of (y-AmNS)UTP, a fluorescent substrate analog to RNApolII. It was observed that OP-treated enzyme showed non-specific substrate recognition, whereas two Zn2+ -native RNApol binds substrate at a single site. Keywords: Reconstitution; 65Zn-blotting; Zinc fingers; CD studies; Abortive initiation 1. Introduction RNA polymerases (RNApol) from different sources have been found to contain Zn2+ ion(s) at a variable stoichiometry. Thus, Escherichia coli RNApol contains 2 mol Zn2+ per mol core enzyme which has a subunit composition of cl,BB’ (Scrutton et al., 1971; Chatterji and Wu, 1982). Metal-substitution experiments and spectroscopic studies suggested that these two Zn2+ are *Corresponding author. Tel. +91 40 672241; Fax +91 40 671195; e-mail: dipan@ccmb.globemail.com 1 Presented at the International Conference on ‘Eukaryotic Expression Vector Systems: Biology and Applications’, National Institute of Immunology, New Delhi, India; 4-8 February 1996. Abbreviations: A, absorbance (1 cm); AmNS, 1-aminonapthalene-5 sulfonate; (y-AmNS)UTP, uridine-5’-triphosphoro-y-1-[5-sulfonic acid] naphthylamidate; CD, circular dichroism; EDTA, ethylenediami- netetraacetic acid; HPLC, high-performance liquid chromatography; OP (or 0. P), o-phenanthroline; PAGE, polyacrylamide gel electro- phoresis; PMSF, phenylmethylsulfonyl fluoride; RNApol, RNA poly- merase(s); RNApolII, RNA polymerase II; SDS, sodium dodecyl sulfate; TFIII, transcription factor III; Trp, tryptophan. 0378-l 119/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO378-1119(96)00710-X not functionally equivalent, one of them lying close to initiation site, so possibly contributing to the active site (Chatterji et al., 1984; Beal et al., 1990) whereas the location of the other is not certain. Zn2+ is found to be associated with highly purified eukaryotic polymerases (Treich et al., 1991). RNApolII from Saccharomy ces cerevisiae was reported to contain 1 g-atom of Zn2+ (Lattke and Weser, 1976). Preliminary experiments car- ried out by one of us in the laboratory of Gunther Eichhorn, National Institute of Health, National Institute of Aging, Gerontology Research Center (Baltimore, MD, USA), showed 2-3 Zn2+ ions per molecule of enzyme where it was purified to homogeneity using HPLC gel filtration and membrane convection chromatography (Butzow et al., 1994). This apparent discrepancy in the number of Zn2+ ions per molecule of enzyme becomes more noticeable when one scans through the reported value of Zn2+ at RNApol from different sources. Wheat germ RNApolII was reported to have 7 g-atom of Zn2+ per molecule of enzyme (Petranyi et al., 1977) whereas that of Euglena gracilis