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Structure of yeast RNA polymerases determined by electron microscopy
ARCHIVES OF BIOCHEMISTRY Vol. 219, No. 1, November,
AND BIOPHYSICS pp. 163-166, 1982
Structure
of Yeast RNA Electron PAULINA
*Laboratorio Znstituto
de Bioquimica and de Ciencim Rioi%gicas,
BULL*
Polymerases Microscopy AND
tLaboratorio Universidad
Received
JORGE
Determined
GARRIDOT,’
de Histologia, Departamento Catdlica de Chile, Casil a
June
by
114-0,
de Biologia Santiago,
Celular, Chile
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 instance, fatty acid synthethase (l), pyruvate carboxylase (2), Escherichia coli RNA polymerase (3), and others (4, 5). In spite of the limitations imposed by the complexity of these systems and of the relative lack of resolution of the method itself, it has been possible to obtain useful information about the overall conformation of these enzymes. Yeast, like other eucaryotes, has three RNA polymerases with different molecular weights and subunit composition (6). These enzymes play a key role in transcription through specific interaction with DNA and other chromatin components of the eucaryotic genome. They are complex enzymes consisting of several polypeptides with sedimentation coefficients of around 16 S (7). It is therefore of considerable interest to determine the shape and possible details of the architecture 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
the molecular weights of the subunits of the enzymes, determined by polyacrylamide gel electrophoresis. METHODS Yeast RNA polymerases I, II, and III were purified to homogeneity according to the procedure of Valenzuela et al. (6). The proteins were diluted to a concentration of 10 pg/ml with 50 mM Tris-HCl (pH 8) containing 0.5 mM EDTA, 0.5 M NaCl, 10 mM 2-mercaptoethanol, 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 200mesh copper grids, and air-dried on filter paper. The staining solutions employed were: 1% many1 acetate (8), 2% ammonium molybdate, pH 8.5, 2% silicotungstate, 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 photogsaphed 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 enlargements 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
be addressed. 163
0003.9861/82/130163-04$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
164
BULL
AND
many1 acetate. Figures la to d show different micrographs of yeast RNA polymerase I stained with uranyl acetate. It can be seen that the preparation is composed of particles of homogeneous size and shape. The images indicate that this enzyme is a globular protein, approximately spherical in shape. The surface is slightly rough and in some molecules, a prominent spike can be detected. In several molecules, it is possible to distinguish a groove, which may represent the separation between the two larger subunits, of molecular weights of 185,000 and 137,000. Except for this, it is impossible to discern the limits between the various subunits. The molecular di-
FIG.
1. Electron
micrographs
of yeast
acetate. (e) A polymerase I preparation ammonium molybdate. (g) Negative stain photungstate on a preparation containing
RNA
GARRIDO
ameters are 12.7 + 0.4 and 11.0 & 1.4 (SD) nm. The complex does not present obvious symmetry, a fact that was expected since the molecular weights of the subunits are all different. In order to confirm the size obtained for the RNA polymerase molecules, ferritin was introduced as an internal marker in enzyme preparations. The molecular diameter of ferritin, 12-13 nm, has been well established by different methods (9). The results of these observations are shown in Fig. le; they show that ferritin is slightly smaller than RNA polymerase I. The difference between both proteins agrees with the value obtained for the latter.
polymerase
I. (a to
d)
Negative
stain
to which ferritin (A) was added. (f) Negative with sodium silicotungstate. (h) Negative stain ferritin (A). The bars represent 50 nm.
with stain with
many1 with phos-
ELECTRON
FIG. 2. Electron micrographs many1 acetate. (b) Polymerase
MICROSCOPY
OF
YEAST
of yeast RNA polymerases III, same negative stain.
The micrographs that follow show the aspect of enzyme I after negative staining with other solutions, such as ammonium molybdate (Fig. If), silicotungstate (Fig. lg), and phosphotungstate (Fig. lh). In the latter preparation, ferritin was also included. It can be seen that although the resolution obtained is lower, the size observed after the different staining solutions is the same. This indicates that the results obtained -are reproducible and do not depend on the technique employed. This observation, especially on the globular nature of the enzyme, allows us to assign to it a molecular weight of 650,000 from the previously determined sedimentation coefficient of 16 S. The results on the molecular diameter permit an estimation of the molecular weight by other means. With the diameter values we have found for yeast RNA polymerase I, 12.7 and 11 nm, a volume 1.33 times larger than that from E. coli RNA polymerase (10) can be calculated assuming equal molecular density. Since the molecular weight of E. coli RNA polymer-
RNA
165
POLYMERASES
II and III. (a) Polymerase The bar represents 50 nm.
II stained
with
ase has been determined to be 480,000500,000 (ll), enzyme I from yeast could have a molecular weight of 640,000665,000, a value in good agreement with that calculated from the sum of the sizes of the individual subunits as determined by polyacrylamide gel electrophoresis and with the molecular weight assigned on the basis of the sedimentation coefficient (7). Figures 2a and b show electron micrographs of RNA polymerases II and III, respectively. These enzymes are also globular, roughly spherical, and of compact appearance. The diameters found for polymerase II are 12.7 f 1 and 12.2 +- 1 (SD) nm and for polymerase III, 13.6 f 0.6 and 11.5 + 1.3 (SD) nm. These results indicate that polymerase II is the most spherical and more compact of the three, while polymerase III is the largest and less regular. ACKNOWLEDGMENTS The authors gratefully acknowledge the stimulating advice of Dr. Pablo Valenzuela, and the outstanding
166
BULL
AND GARRIDO
technical assistance of Lucy Messen. This work was supported by Grants 66/81 and 78/81 from the Direccibn de Investigacibn, Universidad Catblica de Chile and by funds from UNDP/Unesco Program RLA 78/024-20.
7.
REFERENCES 1. WIELAND, F., SIESS, E. A., RENNER, L., VERFURTH, C., AND LYNEN, C. (1978) Pm. Nat.
Acad.
Sci. USA
75,
6.
5792-5796.
2. COHEN, N., BEEGER, H., UTTER, M. F., AND WRIGLEY, N. (1979) J. Biol. Chem. 254, 17401747. 3. FUCHS, E., ZILLIG, W., HOFSCHNEIDER, P. H., AND PREUSS, A. (1964) J. Mol. Biol. 10, 546550. 4. REED, C. J., AND Cox, D. J. (1970) in The Enzymes (Boyer, P., ed.), 3rd ed., pp. 213-240, Academic Press, New York. 5. FINCH, J. T. (1975) in The Proteins (Neurath, A.,
8.
9. 10. 11.
and Hill, R. L., eds.), Vol. I, pp. 413-497, Academic Press, New York. VALENZUELA, P., BELL, G. I., WEINBERG, F., AND RULER, W. J. (1978) in Methods in Cell Biology (Stein, G., and Stein, J., eds.), Vol. XIX, pp. l-26, Academic Press, New York. VALENZUELA, P., WEINBERG, F., BELL, G. I., AND RUTTER, W. J. (1976) J. Biol. Chem. 251, 1464-1470. HASCHEMEYER, R. E. (1970) in Advances in Enzymology (Nord, F., ed.), Vol. 33, pp. 71-118, Interscience, New York. MUNRO, H. N., AND LINDER, N. C. (1978) Physiol. Reu. 58, 317-396. SLAYTER, H. S., AND HALL, C. E. (1966) J. Mol. Biol. 21, 83-114. BURGESS, R. R. (1976) in RNA Polymerase (Losick, R., and Chamberlin, M., eds.), pp. 69-100, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
AND BIOPHYSICS pp. 163-166, 1982
Structure
of Yeast RNA Electron PAULINA
*Laboratorio Znstituto
de Bioquimica and de Ciencim Rioi%gicas,
BULL*
Polymerases Microscopy AND
tLaboratorio Universidad
Received
JORGE
Determined
GARRIDOT,’
de Histologia, Departamento Catdlica de Chile, Casil a
June
by
114-0,
de Biologia Santiago,
Celular, Chile
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 instance, fatty acid synthethase (l), pyruvate carboxylase (2), Escherichia coli RNA polymerase (3), and others (4, 5). In spite of the limitations imposed by the complexity of these systems and of the relative lack of resolution of the method itself, it has been possible to obtain useful information about the overall conformation of these enzymes. Yeast, like other eucaryotes, has three RNA polymerases with different molecular weights and subunit composition (6). These enzymes play a key role in transcription through specific interaction with DNA and other chromatin components of the eucaryotic genome. They are complex enzymes consisting of several polypeptides with sedimentation coefficients of around 16 S (7). It is therefore of considerable interest to determine the shape and possible details of the architecture 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
the molecular weights of the subunits of the enzymes, determined by polyacrylamide gel electrophoresis. METHODS Yeast RNA polymerases I, II, and III were purified to homogeneity according to the procedure of Valenzuela et al. (6). The proteins were diluted to a concentration of 10 pg/ml with 50 mM Tris-HCl (pH 8) containing 0.5 mM EDTA, 0.5 M NaCl, 10 mM 2-mercaptoethanol, 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 200mesh copper grids, and air-dried on filter paper. The staining solutions employed were: 1% many1 acetate (8), 2% ammonium molybdate, pH 8.5, 2% silicotungstate, 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 photogsaphed 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 enlargements 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
be addressed. 163
0003.9861/82/130163-04$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
164
BULL
AND
many1 acetate. Figures la to d show different micrographs of yeast RNA polymerase I stained with uranyl acetate. It can be seen that the preparation is composed of particles of homogeneous size and shape. The images indicate that this enzyme is a globular protein, approximately spherical in shape. The surface is slightly rough and in some molecules, a prominent spike can be detected. In several molecules, it is possible to distinguish a groove, which may represent the separation between the two larger subunits, of molecular weights of 185,000 and 137,000. Except for this, it is impossible to discern the limits between the various subunits. The molecular di-
FIG.
1. Electron
micrographs
of yeast
acetate. (e) A polymerase I preparation ammonium molybdate. (g) Negative stain photungstate on a preparation containing
RNA
GARRIDO
ameters are 12.7 + 0.4 and 11.0 & 1.4 (SD) nm. The complex does not present obvious symmetry, a fact that was expected since the molecular weights of the subunits are all different. In order to confirm the size obtained for the RNA polymerase molecules, ferritin was introduced as an internal marker in enzyme preparations. The molecular diameter of ferritin, 12-13 nm, has been well established by different methods (9). The results of these observations are shown in Fig. le; they show that ferritin is slightly smaller than RNA polymerase I. The difference between both proteins agrees with the value obtained for the latter.
polymerase
I. (a to
d)
Negative
stain
to which ferritin (A) was added. (f) Negative with sodium silicotungstate. (h) Negative stain ferritin (A). The bars represent 50 nm.
with stain with
many1 with phos-
ELECTRON
FIG. 2. Electron micrographs many1 acetate. (b) Polymerase
MICROSCOPY
OF
YEAST
of yeast RNA polymerases III, same negative stain.
The micrographs that follow show the aspect of enzyme I after negative staining with other solutions, such as ammonium molybdate (Fig. If), silicotungstate (Fig. lg), and phosphotungstate (Fig. lh). In the latter preparation, ferritin was also included. It can be seen that although the resolution obtained is lower, the size observed after the different staining solutions is the same. This indicates that the results obtained -are reproducible and do not depend on the technique employed. This observation, especially on the globular nature of the enzyme, allows us to assign to it a molecular weight of 650,000 from the previously determined sedimentation coefficient of 16 S. The results on the molecular diameter permit an estimation of the molecular weight by other means. With the diameter values we have found for yeast RNA polymerase I, 12.7 and 11 nm, a volume 1.33 times larger than that from E. coli RNA polymerase (10) can be calculated assuming equal molecular density. Since the molecular weight of E. coli RNA polymer-
RNA
165
POLYMERASES
II and III. (a) Polymerase The bar represents 50 nm.
II stained
with
ase has been determined to be 480,000500,000 (ll), enzyme I from yeast could have a molecular weight of 640,000665,000, a value in good agreement with that calculated from the sum of the sizes of the individual subunits as determined by polyacrylamide gel electrophoresis and with the molecular weight assigned on the basis of the sedimentation coefficient (7). Figures 2a and b show electron micrographs of RNA polymerases II and III, respectively. These enzymes are also globular, roughly spherical, and of compact appearance. The diameters found for polymerase II are 12.7 f 1 and 12.2 +- 1 (SD) nm and for polymerase III, 13.6 f 0.6 and 11.5 + 1.3 (SD) nm. These results indicate that polymerase II is the most spherical and more compact of the three, while polymerase III is the largest and less regular. ACKNOWLEDGMENTS The authors gratefully acknowledge the stimulating advice of Dr. Pablo Valenzuela, and the outstanding
166
BULL
AND GARRIDO
technical assistance of Lucy Messen. This work was supported by Grants 66/81 and 78/81 from the Direccibn de Investigacibn, Universidad Catblica de Chile and by funds from UNDP/Unesco Program RLA 78/024-20.
7.
REFERENCES 1. WIELAND, F., SIESS, E. A., RENNER, L., VERFURTH, C., AND LYNEN, C. (1978) Pm. Nat.
Acad.
Sci. USA
75,
6.
5792-5796.
2. COHEN, N., BEEGER, H., UTTER, M. F., AND WRIGLEY, N. (1979) J. Biol. Chem. 254, 17401747. 3. FUCHS, E., ZILLIG, W., HOFSCHNEIDER, P. H., AND PREUSS, A. (1964) J. Mol. Biol. 10, 546550. 4. REED, C. J., AND Cox, D. J. (1970) in The Enzymes (Boyer, P., ed.), 3rd ed., pp. 213-240, Academic Press, New York. 5. FINCH, J. T. (1975) in The Proteins (Neurath, A.,
8.
9. 10. 11.
and Hill, R. L., eds.), Vol. I, pp. 413-497, Academic Press, New York. VALENZUELA, P., BELL, G. I., WEINBERG, F., AND RULER, W. J. (1978) in Methods in Cell Biology (Stein, G., and Stein, J., eds.), Vol. XIX, pp. l-26, Academic Press, New York. VALENZUELA, P., WEINBERG, F., BELL, G. I., AND RUTTER, W. J. (1976) J. Biol. Chem. 251, 1464-1470. HASCHEMEYER, R. E. (1970) in Advances in Enzymology (Nord, F., ed.), Vol. 33, pp. 71-118, Interscience, New York. MUNRO, H. N., AND LINDER, N. C. (1978) Physiol. Reu. 58, 317-396. SLAYTER, H. S., AND HALL, C. E. (1966) J. Mol. Biol. 21, 83-114. BURGESS, R. R. (1976) in RNA Polymerase (Losick, R., and Chamberlin, M., eds.), pp. 69-100, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.