Restoration of binding of oxidized transcription factor m A to 5 S RNA

7S particles from Xenopus oocytes were completely dissociated under nonreducing conditions. Studies using glycerol gradient centrlfugation show that unlike the native 7S particle in which 5S RNA and TFIIIA co-sedimented in a fairly sharp peak,the RNA from the denatured 7S sedlmented at the position corresponding to the 5S RNA and the TFIIIA sedimented as a wide peak between 6S and 12S. Thioredoxin froo E.coli can catalyze the reactivation of the TFIIIA as measured by its ability to reform the 7S particle . The rate of reactivation with thioredoxin was significantly greater than with dlthiothreltol. Oxidized thioredoxin was unable to reactivate TFIIIA. Pure TFIIIA can be inactivated and subsequently reactivated in the same way by formation of a cross-linked structure via intermolecular disulfide bridges. INTRODUCTION Xenopus laevis factor III A (TFIIIA) is a positive transcription factor that binds to an lntragenic control region of the 5S RNA gene , promoting its transcription by RNA polymerase III (1-4). TFIIIA associates with 5S RNA, and in immature oocytes is found in the form of a 7S nucleoprotein complex (5). The ability of TFIIIA to bind either the 5S RNA gene or its product led to the suggestion that It might have a potential role in feedback regulation of 5S gene expresslon(6). Besides the specific binding to the 5S RNA gene, TFIIIA possesses other multiple activities such as a DNA-dependent ATPase activity (7), the promotion of the reassociation of complementary single stranded DNA (8) and the induction of dynamic chromatln assembly on cloned Xenopus 5S RNA genes (9). TFIIIA Is a Zn binding protein (10,11) which contains nine tandem repeats of a 30 amlno acid cluster having two cysteines and two hlstidines in each repeat. This observation led to the suggestion that each repeat binds one atom of Zn. In this work we present evidence that one possible route of inactivation of TFIIIA, either in pure preparations or as a part of the 7S nucleoprotein particle, is via oxidation and subsequent formation of an heterogeneous © IRL Press Limited, Oxford, England. 8497 Nucleic Acids Research population of molecules crosslinked by dlsulfides bonds. This inactivation can be reverted by the action of thioredoxin from E.coli. MATERIALS AND HETHODS Immature Xenopus laevls frogs were obtained from Nasco. Bio Rex 70 and Bio Gel HT were purchased from Bio Rad, and DEAE cellulose from Whatman. Thioredoxin was from Chemical Dynanics Corp. Purification of the 7S RNP particle 7S RNP particles were isolated using a modification of the procedure of Hanas (12). Briefly, 7S particles Isolated by glycerol gradient centrifugation were loaded onto a Bio Rex 70 column equilibrated with 30 mM Hepes, pH 7.5, 0.025 M KC1, 1.5 mM MgCl2, 0,5 mM DTT and 5% glycerol (5-10 mg/ml packed resin). Flow-through fractions were collected and loaded on a column of DEAE-cellulose. The column was washed and eluted with the sane buffer containing 0.2 M KC1 and 0.32 M KC1 respectively. The 7S particle fraction was adjusted to 30 % glycerol and less than 0.16 M KC1. The protein concentration was between 1-2 mg/ml as measured by the method of Bradford (13) using bovine serum albunin as a standard and more than 90% pure as judged by SDS-PAGE (14). This complex was stable at -20°C for more than one year as determined by agarose gel electrophoresis( see below). Preparation of TFIIIA TFIIIA was isolated by loading purified 7S RNP particles onto a Bio Gel HT column ( 5 mg of protein/nl packed resin ) equilibrated In 10 mM phosphate buffer pH 7.5, 25% glycerol. The column was washed with 10 mM phosphate buffer pH 7.5, 25 mM Hepes pH 7.5, 0.5 mM DTT, 0.1 M KC1 and the free TFIIIA vas eluted with the same buffer containing 1 M KC1. The TFIIIA-containing fraction was adjusted to 50% glycerol and 0.5 M KC1 and stored at -70°C. We found this method particularly suitable to study TFIIIA inactivation by alkaline buffer dilutions (see below), since it yielded highly concentrated TFIIIA. Preparation of 5S RNA 5S RNA was purified using an extension of the procedure of Dignam et al. (15) for the purification of TFIIIA. Following elution of TFIIIA from the DEAE-cellulose with urea, the column was washed with 10 mM Tris-HCl pH 7.5, 0.5 mM EDTA and then 5S RNA was eluted with the same buffer containing 0.5 M KC1. The eluted sample was dlalyzed against 10 mM Trls-HCl pH 7.5 for 2 hours, followed by extraction with phenol-CHCl3 (1:1).then with CHCI3 and finally precipitated with ethanol.