ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 219, No. 1, November, pp. 163-166, 1982 Structure of Yeast RNA Polymerases Determined by Electron Microscopy PAULINA BULL* AND JORGE GARRIDOT,’ *Laboratorio de Bioquimica and tLaboratorio de Histologia, Departamento de Biologia Celular, Znstituto de Ciencim Rioi%gicas, Universidad Catdlica de Chile, Casilla 114-0, Santiago, Chile Received June 14, 1982 Methods have been developed for the examination of yeast RNA polymerases I, II, and III by electron microscopy. The results enabled us to establish the size and shape of a eucaryotic RNA polymerase for the first time. The enzymes are roughly spherical in shape and compact in appearance. Their measured molecular diameters are 12.7 + 0.4 and 11.0 f 1.4 (SD) nm for polymerase I, 12.7 + 1.1 and 12.2 + 1.0 (SD) nm for polymerase II, and 13.6 f 0.6 and 11.5 + 1.3 (SD) nm for polymerase III. There are several examples of enzymes and enzymatic complexes that have been studied by electron microscopy, for in- stance, fatty acid synthethase (l), pyru- vate carboxylase (2), Escherichia coli RNA polymerase (3), and others (4, 5). In spite of the limitations imposed by the com- plexity of these systems and of the relative lack of resolution of the method itself, it has been possible to obtain useful infor- mation about the overall conformation of these enzymes. Yeast, like other eucary- otes, has three RNA polymerases with dif- ferent molecular weights and subunit com- position (6). These enzymes play a key role in transcription through specific interac- tion with DNA and other chromatin com- ponents of the eucaryotic genome. They are complex enzymes consisting of several polypeptides with sedimentation coeffi- cients of around 16 S (7). It is therefore of considerable interest to determine the shape and possible details of the architec- ture of these molecules. The determination of the axial ratios is also important, since it could result in a better correlation of the known sedimentation coefficients with the molecular weights obtained by the sum of i To whom all correspondence should be addressed. the molecular weights of the subunits of the enzymes, determined by polyacryl- amide gel electrophoresis. METHODS Yeast RNA polymerases I, II, and III were purified to homogeneity according to the procedure of Val- enzuela et al. (6). The proteins were diluted to a con- centration of 10 pg/ml with 50 mM Tris-HCl (pH 8) containing 0.5 mM EDTA, 0.5 M NaCl, 10 mM 2-mer- captoethanol, and 20% glycerol. Carbon films prepared on mica substrates were floated on the protein solutions for 5 min, transferred to the staining solution for l-5 min, picked up on 200- mesh copper grids, and air-dried on filter paper. The staining solutions employed were: 1% many1 acetate (8), 2% ammonium molybdate, pH 8.5, 2% silico- tungstate, pH 5, and 2% phosphotungstate, pH 7. The specimens were examined in a Siemens Elmiskop 102 at accelerating voltages of 80 and 100 kV and pho- togsaphed at a direct magnification of 80,000. The magnification was calibrated by means of a 54,800. lines-per-inch grating replica. Measurements were made both on the negatives and on paper enlarge- ments by means of a calibrated optical magnifier. RESULTS AND DISCUSSION Several negative staining techniques were tried on yeast RNA polymerases which had been purified to homogeneity (6). The best results were obtained with 163 0003.9861/82/130163-04$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.