Proc. Natl. Acad. Sci. USA Vol. 88, pp. 9984-9988, November 1991 Biochemistry Zinc transfer from transcription factor IIIA fingers to thionein clusters (metallothionein/zinc fingers/zinc clusters) JIN ZENG*, BERT L. VALLEEtt, AND JEREMIAS H. R. KAGI*t *Biochemisches Institut der Universitat Zurich, Winterthurerstrasse 190, CH-8057, Zurich, Switzerland; and tCenter for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, 250 Longwood Avenue, Boston, MA 02115 Contributed by Bert L. Vallee, August 12, 1991 ABSTRACT The rapid induction of thionein (apometal- for zinc are comparable in magnitude to those of zinc lothionein) by many endogenous stimuli such as steroid hor- metalloproteins, the rates at which they transfer metals upon mones, cytokines, and second messengers suggests that this exposure to chelating agents (4) and exchange metals among cysteine-rich, metal binding protein participates in an as yet metallothionein isoforms (5, 6) are much higher. This kinetic undefined role in cellular regulatory processes. This study lability of the zinc thiolate clusters allows them to play a demonstrates with DNA and RNA binding assays and in vitro dynamic metabolic role in which thionein and its conjugate transcription measurements that thionein suppresses the bind- zinc metallothionein serve as intracellular distributors and ing of the Xenopus laevis zinc finger transription factor IDlA mediators of zinc wherever and whenever needed for what- (TFIilA) to 5S RNA and to the 5S RNA gene and abrogates the ever purpose (7). Thus, the thionein/metallothionein couple capacity of TFIIIA to initiate the RNA polymerase III- may be viewed as a collation/allocation system, regulating catalyzed synthesis of 5S RNA. The effect is reversed by the the availability of zinc pivotal to multiple and diverse func- addition of zinc and is not observed in the TFIHA-independent tions on demand (8). Based on its unusual composition and transcription of a tRNA gene by the same RNA polymerase. In structure, the participation of metallothionein in embryonic view of the strong tendency of thionein to complex posttran- development, cellular differentiation, and the transmission of sition metals such as zinc, one effect of its enhanced synthesis genetic information and cell proliferation was anticipated and in vivo could be to reduce the intracellular diosability of zinc predicted long before the relevant biochemistry became and thus modulate the actions of zinc-dependent enzymes and known and understood (9). proteins, most notably those of the zinc finger transcription A number of studies have demonstrated that some apoen- factors. zymes can acquire zinc from metallothionein under in vitro conditions (10), but the in vivo significance of this phenom- Zinc is a constituent of many proteins and enzymes and as enon has not been confirmed. It is of interest that although such is indispensable to catalysis, gene expression, and metallothionein does not donate its zinc to the zinc-depleted intracellular signaling. This functional variability has been form of the vertebrate zinc finger transcription factor Spl attributed to its versatile chemistry, which, though seemingly (11), an excess of the apoprotein thionein can abstract zinc prosaic, turns out to be ideally suited for deployment in from the native, zinc-containing form of Spl, which conse- biomacromolecular systems to subserve widely different quently loses its capacity to bind to its cognate DNA and to biological processes. One of these properties is the stability activate downstream gene transcription by RNA polymerase of the Zn(II) oxidation state in a range of biological media, 11 (12). another is the multiplicity of its coordination number, and a The present study demonstrates that thionein also removes third is its several and adaptable coordination geometries. zinc from the Xenopus laevis zinc finger protein transcription Together these combine to generate a remarkably flexible factor IIIA (TFIIIA) in an analogous manner, thereby inter- spectrum ofchemical affinities and reactivities (1). At present fering with its functional properties. TFIIIA has been re- four major classes of zinc proteins can be distinguished: the ported to contain from two to nine zinc atoms per molecule >300 enzymes whose catalytic activity or structural integrity (13, 14), a variation that could be physiological or analytical. requires the presence of zinc, the seemingly innumerable The following data show that increased thionein biosynthesis zinc-dependent transcription factors that regulate long- and could suppress the binding ofTFIIIA to its cognate DNA and short-term gene expression, the glucocorticosteroid and to 5S RNA as well as its activating effect on 5S RNA gene other receptors, and the zinc cluster-bearing metallothio- transcription by RNA polymerase III. neins (1, 2). The metallothioneins are ubiquitous proteins that were discovered almost contemporaneously with the first zinc MATERIALS AND METHODS enzymes, but a precise understanding of their biological Cell Line. HeLa cells were maintained in Joklik modified role(s) has yet to be achieved. The mammalian forms contain minimal essential culture medium at 370C. 61 amino acid residues, among them 20 cysteines that bind a Plasmids. The construction of 5S RNA and tRNA genes total of seven zinc (or cadmium) ions in two thiolate clus- has been described (15, 16). ters-i.e., Zn3Cys9 and Zn4Cysl1-located within the amino- Purification of 7S Ribonucleoprotein (7S RNP) and TFIA. and carboxyl-terminal domains, respectively (2). These The 7S RNP was prepared from immature X. laevis oocytes structures, for which there are no appropriate inorganic by glycerol gradient centrifugation and DEAE-cellulose models, combine high thermodynamic stability of metal chromatography (17, 18). TFIIIA (Mr 38,600) was separated binding with high kinetic lability of metal exchange, which from 5S RNA by ammonium sulfate precipitation and quan- may be characteristic. Thus, although their binding constants tified by the Bradford method with bovine serum albumin as The publication costs of this article were defrayed in part by page charge Abbreviations: TFIIIA, transcription factor IIIA; 7S RNP, 7S ribo- payment. This article must therefore be hereby marked "advertisement" nucleoprotein; ICR, internal control region of the 5S RNA gene. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 9984 Downloaded by guest on September 30, 2021 Biochemistry: Zeng et al. Proc. Natl. Acad. Sci. USA 88 (1991) 9985 a standard (19). TFIIIA is assumed to bind nine zinc ions to its transcription product, 5S RNA (13, 14). Thionein is one maximally. of the most potent naturally occurring metal binding agents Preparation of Metaflothionein and Thionein. (Cd,Zn)7- and, hence, its effect on the interaction of this zinc finger metallothionein was purified from the livers of rabbits in- protein with its cognate nucleic acids was investigated by jected with CdCl2; Zn7-thionein was isolated from human means of a gel retardation assay. The 50-bp ICR DNA liver (20). Thionein (Mr 6100) was prepared from metallothio- segment ofthe 5S RNA gene (22) was synthesized chemically nein by acidification with HCO and removal ofthe metal by gel and used as a TFIIIA binding site subsequent to 32P end- filtration in 10 mM HCI (21). Immediately before use, thio- labeling. The TFIIIA-DNA complex is found as a retarded nein was neutralized with 0.1 M Tris HCI (pH 8.0). band in polyacrylamide gel electrophoresis (Fig. 1A, lanes 1 Preparation of SS RNA Gene Deoxyoligonucleotide Probe. A and 2). To detect whether or not thionein inhibits the binding 50-base-pair (bp) deoxyoligonucleotide with the sequence of of TFIIIA to DNA, TFIIIA and thionein were mixed in the internal control region ofthe 5S RNA gene (ICR) (22) was binding buffer supplemented with 9 ,uM ZnCl2 and incubated synthesized with an Applied Biosystems DNA synthesizer. for 20 min before the addition of labeled ICR. Fig. 1A (lane After deprotection in ammonium hydroxide and lyophiliza- 7) shows that as little as 4 4M thionein nearly completely tion the DNA segment was purified by electrophoresis on a prevents 300 nM TFIIIA from binding to the DNA probe. 12% sequencing gel. Moreover, the addition of Zn(II) reverses the interference of Gel Retardation Assays for Binding of TFIIIA to DNA. The thionein with TFIIIA binding to ICR (Fig. 1B, lane 4). 32P end-labeled ICR deoxyoligonucleotide probe was incu- Thionein Suppresses Interaction of TFIIA with 5S RNA to bated with -200 ng ofTFIIIA for 20 min at room temperature Form a 7SRNP Complex. TFIIIA is known to bind to 5S RNA in an 18-/.l volume containing 30 mM Tris HCI (pH 7.5), 70 in a zinc-dependent manner (25) to form 7S RNP. Fig. 2A mM KCl, 2 mM MgCl2, 0.5 mM dithiothreitol, 6% glycerol, with 9 0.1% Nonidet P-40, 2 pug of poly(dI-dC), and 9AM ZnC12, the (lane 6) shows that in a binding buffer supplemented last added to activate TFIIIA as previously suggested (14). ,uM ZnC12, 4tuM thionein also abolishes the binding of300 nM Complex formation with TFIIIA was monitored by the re- TFIIIA to the RNA. Further, addition of zinc reverses this tardation of the movement of the DNA probe on electropho- effect (Fig. 2B, lane 4). Significantly, incubation of Zn7- resis in a 6% native polyacrylamide gel run in 89 mM Tris thionein with TFIIIA does not affect its binding to either borate buffer (pH 8.3) without EDTA. Gels were dried and DNA or RNA (Fig. 1A, lane 10; Fig. 2A, lane 10). exposed to x-ray film. To study the effects of thionein or of Thionein competition with TFIIIA for zinc is energetically Zn7-thionein on TFIIIA-DNA complex formation prior to feasible; the average apparent association constant for zinc in incubation with the ICR probe, TFIIIA (final concentration, Zn7-thionein, at pH 7, is about 2 x 1012 M-1 (21) and around 300 nM) was preincubated with thionein or Zn7- coincides with the minimum value of 1012 M-1 estimated for thionein at room temperature for 20 min.