- Email: [email protected]
[38] The characterization of ribosomal subunits of eukaryotes by ultracentrifugal techniques. Criteria of purity
[38]
RIBOSOMAL SUBUNITS OF EUKARYOTES
387
In this article, we have presented a detailed procedure for the preparation of eukaryotic ribosomes and ribosomal subunits. This procedure has been successful with polysomes from all cells and tissues with which we have dealt. Thus, these methods should be effective with ribosomes obtained from other eukaryotic sources. Processed ribosomes are severely depleted of initiation factors 2a-2° and endogenous messenger R N A activity, but will participate in translation with high efficiency when supplemented with these substances.Thus, the procedures which we have described are useful for preparing in vitro amino acid incorporation systems which can be used as sensitive assays for messenger RNA, tRNA, elongation factors, initiation factors, 2"-2e and other components of the protein biosynthetic apparatus which are usually associated with ribosomes prepared by other techniques. Acknowledgments We thank Ms. Marcia Witte for her excellent technical assistance and Demler Farms for the donation of adult Shaver Starcross Leghorn chickens.
[38]
The Characterization of Ribosomal Subunits Eukaryotes by Ultracentrifugal Techniques. Criteria of Purity
of
B y MARY G. HAMILTON
The problem of establishing homogeneity for a complicated structure like a ribosome is not easily solved, Perhaps such a concept should be irrevelant. The subunits of eukaryotic cytoplasmic ribosomes, in their "active" state, seem to behave more like supramolecular complexes than simple ribonucleoproteins.' Moreover, if heterogeneity exists among the population of small subunits of Escherichia coli ribosomes,'-' it probably exists for eukaryotic ribosomes as well. Despite these reservations it is important to try to characterize the subunits as fully as possible. Fortunately, techniques are now available for the complete characterization of ribosomal subunits.:' One can separate subunits by zonal centrifugation," although pressure-induced dissociation M. G. Hamilton, A. Pavlovec, and M. L. Petermann, Biochemistry 10, 3424 (1971). " C. G. Kurland, Annu. Rev. Biochem. 41, 377 (1972). ~M. G. Hamilton and M. E. Ruth, Biochemistry 8, 851 (1969). 'P. S. Sypherd and J. Wireman, this volume [35]; C. C. Sherton et al., this volume [36].
388
RIBOSOME STRUCTURE AND FUNCTION
~v 2
o O
v v v v Z
F~
Z
rnB
[38]
[38]
RIBOSOMAL SUBUNITS OF EUKARYOTES
389
may sometimes complicate the separation. 4~' The subunits can be fixed with formaldehyde to prevent degradation. This is useful in general but essential for the buoyant density measurements which can be used for an estimate of protein content as well as purity. The ultraviolet absorption optical system of the analytical ultracentrifuge permits measurements at very low concentrations. With only a few A260 units one can measure sedimentation coefficients and buoyant densities, and if the solutions are monodisperse, the molecular weight can be measured directly by equilibrium centrifugation, one of the few absolute methods. The table reproduces the results of such measurements on the subunits of rat liver ribosomes. Tashiro and Yphantis ~ and Hill et al. ~' have also used analytical ultracentrifugation to measure molecular weights of ribosomes and subunits. Criteria of P u r i t y : Sedimentation Analysis and B u o y a n t Density M e a s u r e m e n t s Although monodispersity of size is measurable by sedimentation analysis, 7 with ribosomal subunits a single sedimenting boundary may represent more than one species. Ribosomal subunits can both open up to sediment more slowly or dimerize to sediment faster. By some natural perverseness the opened large forms of rat liver ribosomes 8 sediment at almost exactly the same rate as active small subunits and, v i c e versa, dimerized active small ones sediment at almost exactly the same rate as large ones?' One simple test for such cross-contamination is to examine the sample after E D T A treatment. TM This converts the active forms to inactive forms of lower sedimentation (s) rates but has no effect on the "opened" forms. Examples of these cases are illustrated in Figs. 1 and 2 for fractions taken from zonal separations of ribosomes dissociated by the urea method of Petermann and Pavlovec. ~ In Fig. 1 small subunit dimers cosedimenting with the large subunits (upper left) are revealed by E D T A treatment as a 30 S boundary (lower left). In Fig. 2, an opened 41 S form of the large subunit cosedimenting with the small subunits (upper left) is unchanged by E D T A treatment (lower left), but the active 40 S small subunits now sediment at 28 S. Because the buoyant density in CsCI depends on the protein content 4,, R. Baierlein and A. A. Infante, this volume [33]. 5 y. Tashiro and D. A. Yphantis, J. Mol. Biol. 11, 174 (1965). "W. E. Hill, G. P. Rossetti, and K. E. Van Holde, J. Mol. Biol. 44, 263 (1969). 7R. Trautman, S. P. Spragg, and H. B. Halsall, Anal. Biochem. 28, 396 (1969). M. L. Petermann, A. Pavlovec, and M. G. Hamilton, Biochemistry 11, 3925 (1972). "M. G. Hamilton, unpublished observations, 1971. ~°M. L. Petermann, this series, Vol. 20, p. 429. 11M. L. Petermann and A. Pavlovec, Biochemistry 10, 2770 (1971).
390
RIBOSOME STRUCTURE AND FUNCTION
[38]
"~.7 S 15-
1.58 A
162
__.../299S
~
/
9.4S
FIG. 1. Large-subunit fraction from a zonal separation of rat liver ribosomes dissociated by treatment with 2 M urea. Comparison by sedimentation velocity (left side) and isodensity equilibrium centrifugation (right side) of samples fixed with H C H O before (upper row) and after (lower row) E D T A treatment. The patterns were obtained by densitometry of U V films.
152 1.53cj/ml
J
1.64
~40.5S
1.54
J'~" 28.4S > FIG. 2. Small subunit fraction from a zonal separation of rat liver ribosomes dissociated by urea. Upper patterns, control. Lower patterns, after E D T A treatment. See legend of Fig. 1. The patterns on the left were obtained by the photoelectric scanner, and the right-hand ones were obtained by densitometry of U V films.
[38]
RIBOSOMAL SUBUNITS OF EUKARYOTES
391
of the subunit and the active forms differ in protein content (see the table), another criterion of homogeneity is available. This analysis reveals crosscontamination of subunits even more dramatically than does the sedimentation pattern. Figure 1 shows a large subunit fraction examined in this way. While the sedimentation pattern shows mainly a single boundary (upper left), the isodensity pattern (upper right) shows three bands which correspond to small subunits, large subunits, and an intermediate band, which may represent residual ribosomes. Again, E D T A treatment (lower right) corroborates the existence of a mixture. The small subunit does not lose protein after E D T A treatment unless it is physically separated from the reaction mixture. ~' When fixed immediately after the addition of EDTA, it retains its low buoyant density. This is a convenience in this assay since it remains distinguishable from the large subunit, as is shown in Figs. 1 and 2, lower right panels. Technical Details The procedures for the main variations of analytical ultracentrifugation have been fully described in this series 1~ and elsewhere. 13 Chervenka" gives a simplified and practical guide. The calculations, with statistical treatment of the data, have been programmed for a desk-top computer? '~ We use Dr. Trautman's programs for the Olivetti Underwood Programma 101 Desk-Top Computer. Velocity sedimentation, in a density gradient with suitable markers of known s rates, and isodensity equilibrium centrifugation can be performed in preparative ultracentrifuges. Molecular weight measurements by classical equilibrium centrifugation, however, require an analytical ultracentrifuge. A minimum of 2 A~60 units, at a concentration of 0.6-0.8 A260 units/ml, is required for the measurements. Naturally, one would like more material for replicate analyses. Preparation o] Sample. Fixation with formaldehyde, which is necessary for the buoyant density measurement, is also useful in preserving the structure for the lengthy measurements. Buffers such as triethanolamine-HC1 (TEA-HC1), which lack primary amino groups, should be used rather than Tris. HC1. Fractions from the sucrose gradient separation are dialyzed overnight against 2 M formaldehyde, 10 mM TEA.HC1, pH 7.4, 0.2 mM 1, H. K. Schachman, this series, Vol. 4, p. 32; J. Vinograd, Vol. 6, p. 854. 13R. Trautman and M. G. Hamilton, in "Principles and Techniques in Plant Virology" (C. I. Kado and H. O. Agrawal, eds.), p. 491. Van Nostrand-Reinhold, Princeton, New Jersey, 1972. 1, C. H. Chervenka, "A Manual of Methods for the Analytical Ultracentrifuge." Spinco Division of Beckman Instruments, Inc., Palo Alto, California, 1969. 15R. Trautman, Ann. N.Y. Acad. Sci. 164, 52 (1969).
392
RIBOSOME STRUCTURE AND FUNCTION
[38]
MgCI2, and for 1 or 2 days against at least two lots of 0.1 M HCHO, 30 mM KCI, 10 mM TEA-HC1, pH 7.4, 0.2 mM MgC12 (FTKM), a suitable solvent for all the centrifugation studies. If necessary, the sample can be concentrated to 0.6-0.8 A260 units/ml by vacuum dialysis in a SchleicherSchuell collodion bag. EDTA Test for Cross-Contamination o[ Zonal Fractions. An excess of EDTA, 0.1 or 0.2 volumes of 0.1 M Na._, EDTA (depending on the MgC1._, content of the separation buffer), which has been adjusted to pH 7 with NaOH, is added to a 2 A_~60unit aliquot. After a few minutes the sample is dialyzed overnight against buffered 2 M HCHO without MgCle, but switched to FTKM for the final dialysis. Sedimentation Velocity. These runs should be performed first because the samples can be recovered after thorough mixing of the cell contents. The ultraviolet absorption optical system should be used preferably with the photoelectric scanner optics, but the film system can also be used. Multicell runs save time and improve accuracy by eliminating the temperature variable when comparisons of s rates are required. The FTKM buffer has a density of 1.0028 g/ml and a relative viscosity of 1.024. For this solvent and a partial specific volume, ~, of 0.63, the "salt correction" for calculating12,1' sedimentation in water, after the temperature correction to 20 ° has been made, is 1.03. Isodensity Equilibrium Centrilugation. These measurements have been described, a6 We now add 0.1 volume of 0.1 M NaH2PO4, 0.1 M Na2H PO4 to ensure a constant pH near 7.5.17 Also, the higher speeds available in a titanium rotor, 48,000 or 52,000 rpm, are advantageous in increasing the gradients and thus displaying more species. When small subunits are present, an initial density of 1.58 g/ml is useful. Classical Equilibrium Centrilugation. The requirements for monodispersity and lack of cross-contamination must be stressed. Since the high speed method is must be used (the present-day centrifuges are reputed to be unreliable at the lowest available speeds), complications due to the presence of heterogeneity should be avoided. The principles and practice of molecular weight measurements have been concisely described by Van Holde? 9 The photoelectric scanner optics must be used for UV measurements. The runs are made in the cold in the An-J rotor in multichannel cells as equipped with sapphire windows. No oil base is used. The solvent side ~6M. ~TH. D. 19K. of
G. Hamilton, this series, Vol. 20, p. 512. A. Wood, Virology 43, 511 (1971). A. Yphantis, Biochemistry 3, 297 (1964). E. Van Holde, "Sedimentation Equilibrium." Fractions I, p. 1. Spinco Division Beckman Instruments, Inc., Palo Alto, California, 1969.
[38]
RIBOSOMAL SUBUNITS OF EUKARYOTES
393
holds 0.13 ml of dialyzate and the sample side 0.10 ml of a solution of about 0.6 A._,6,, units/ml. Nine samples can be run at one time, and conveniently over the weekend; this is sufficient time to reach "practical" equilibrium, i.e., no change in the pattern with time. Figure 3 provides a guide for the selection of speed for the equilibrium run. This has been calculated from an equation given by Yphantis '~ for the desired concentration distribution. Reestablishing equilibrium at several higher speeds, e.g., 20% and 40% higher, improves precision by providing additional data. This usually takes an additional 24 hours for each new speed, and by clearing the meniscus area also provides baselines that can be used for the lower speeds. Establishment of the correct baseline is a problem with the high speed method. The solution columns can be scanned at 265 nm and 280 nm at the slowest scan speed to magnify the x dimension, x is converted to radius, r, in the usual way."-"" The y dimension is converted to absorbancy, c, by means of the calibration "steps" which accompany each scan. TM Since the absorbance at 280 nm is about half that at 260 nm, the 280 nm readings provide an optical dilution for the region near the base of the solution column. The data should be plotted on graph paper as log c vs. r-' and inspected for linearity. Frequently, points near the top of the solution column show a distinct curvature. This may indicate a poor choice of baseline, although heterogeneity can give a positive curvature and nonideality a negative one to the line. Readings less than 0.1 A unit probably should be discarded. Although the deficiencies of the log c vs. r ~ plot
15,000
-
I0,000c:::, 7,0005,000a:: 4,0000 5,000O3
2,000 -
1,000
0.1
012 0.[3 014015 017 I'.0 ]15 210 3'.0 41.050 7'0 MOL. WT x 10-6
FIG. 3. Graph for selection of speed for high speed equilibrium centrifugation of a ribonucleoprotein with ~ = 0.63, in a buffer with a density of 1.002 g / m l in a 3 mm column at 5 °. Adapted from K. E. Van Holde, "Sedimentation Equilibrium." Fractions 1, p. 1. Spinco Division, Beckman Instruments, Inc., Palo Alto, California, 1969.
394
RIBOSOME STRUCTURE AND FUNCTION
[38]
as an index of homogeneity have been noted, '-'° it is the simplest method to apply to scanner data. In the equation 4.606RT d(log c) (1 -- ~p)o~2 dfr 2)
Mw-
Mw is the weight-average molecular weight; R, the gas constant (8.313 X 10 r ergs deg-1 mol-0; T, the absolute temperature; p, the solution density (the solutions are so dilute that the solvent density of 1.002 g/ml may be used); ~, the partial specific volumeZl; % the angular velocity (2 rr rpm/60); and [d(log c)]/d(r'-') is the slope of the line obtained from the plot of log c vs. r -°. We have found ~2 that replicate measurements are essential for precision with the UV optical system (see also the table). When more material is available, the molecular weight measurements should be made with the interference optical system. This requires about 0.1 ml of a solution containing 4-6 A._,60units/ml. Plate reading is laborious (unless automated)~2% but more detailed and precise calculations of the molecular weight distribution can be madeY 3 Double exposures with schlieren optics, -~4 after masking of the interference image, provide additional data ( d c / d r ) for a plot of
the slope of which is proportional to M:, the Z-average molecular weight in the equation
Mz =
(1
-- ~p)o~2-
d(r ~)
Identity of Mw and M: is an indication of homogeneity. C o m m e n t s on O t h e r M e t h o d s
Obviously, with the chemical composition from the buoyant densityTM and the size of the RNA molecule, one can estimate the molecular weight of a ribosomal subunit. The molecular weight of RNA can be measured ~°H. Fujita and J. W. Williams, J. Phys. Chem. 70, 309 (1966). 21The partial specific volume can be estimated from the composition of the particle. In the expression, ~ = 0RNA( % R N A / 1 0 0 ) + ~protei,~ (%protein/100), ~a~.~ is 0.53 (measured pycnometrically for N a R N A ) and O~,~ot~,, is 0.74 (calculated from the amino acid composition). For rat liver ribosomal subunits this value coincides with the reciprocal of the buoyant density in CsC1, but for other ribosomes it may not. :ZM. G. Hamilton, Biochim. Biophys. Acta 134, 473 (1967). 2:~ Photographic labeling of fringes as suggested by S. J. Edelstein and G. H. Ellis [Analytical Biochemistry 43, 89 (1971)] helps in plate alignment on the microcomparator. :3D. C. Teller, T. A. Horbett, E. G. Richards, and H. K. Schachman, Ann. N.Y. Acad. Sci. 164, 66 (1969). 2~C. H. Chervenka, Anal. Chem. 38, 356 (1966).
[39]
HYDROGEN-TRITIUMEXCHANGE STUDIES ON RIBOSOMES
395
by sedimentation equilibrium,'-"-' but this requires purified RNA. A simple treatment with the detergent sodium dodecyl sulfate releases the R N A for examination by electrophoretic analysis in polyacrylamide gels or sedimentation analysis. Both methods can reveal the cross-contamination of large and small subunits discussed above, but obtaining the molecular weight by these methods may be a problem. The gel technique depends on calibration with molecules of known size and conformation and may be in serious error/'~ Estimating molecular weight from sedimentation velocity studies is also dependent on calibration curves or assumptions about shape and hydration. Sedimentation in formaldehyde is not the answer for eukaryotic ribosomal RNA's,',='" although it seems to work for bacterial ones and viral RNA's/G,'-'z A new electrophoretic technique using a nonaqueous solvent may prove useful. =~ ~-~I. B. Dawid and J. W. Chase, J. Mol. Biol. 63, 217 (1972). :" M. L. Fenwick, Biochem. J. 107, 851 (1968). "~H. Boedtker, J. Mol. Biol. 35, 61 (1968). ~ D. Z. Staynov, J. C. Pinder, and W. B. Gratzer, Nature (London) 235, 108 (1972).
[39]
Hydrogen-Tritium
Exchange
Studies on Ribosomes 1
B y MELVIN V. SIMPSON
The method of hydrogen-tritium exchange ( H X ) has been used in the study of some of the conformational properties of ribosomes/-s The method is very suitable for detecting changes in ribosomal conformation, particularly those which occur while the ribosome is functioning2 -7 Reviews on the theoretical aspects as well as the experimental aspects of H X have appeared 9,1° as well as a detailed description and discussion by 1Much of the research upon which this article is based was supported by Grants Nos. GB 5597 and GB 8375 from the National Science Foundation. -"L. A. Page, S. W. Englander, and M. V. Simpson, Biochemistry 6, 968 (1967). 3M. I. Sherman and M. V. Simpson, Cold Spring Harbor Syrup. Quant. Biol. 34, 220 (1969). ' M. I. Sherman, D. Chuang, and M. V. Simpson, Cold Spring Harbor Syrup. Quant. Biol. 34, 109 (1969). ~M. I. Sherman and M. V. Simpson, Proc. Nat. Acad. Sci. U.S. 64, 1388 (1969). '~D. Chuang, H. A. Silberstein, and M. V. Simpson, Arch. Biochem. Biophys. 144, 778 (1971). 7D. Chuang and M. V. Simpson, Proc. Nat. Acad. Sci. U,S. 68, 1474 (1971). M. I. Sherman, Eur. J. Biochem. 25, 291 (1972). ~A. Hvidt and S. O. Nielson, Advan. Protein Chem. 21, 287 (1966). :0 S. W. Englander, in "Poly-a-Amino Acids: Protein Models for Conformational Studies" (G. Fasman, ed.), p. 339. Dekker, New York, 1967.
RIBOSOMAL SUBUNITS OF EUKARYOTES
387
In this article, we have presented a detailed procedure for the preparation of eukaryotic ribosomes and ribosomal subunits. This procedure has been successful with polysomes from all cells and tissues with which we have dealt. Thus, these methods should be effective with ribosomes obtained from other eukaryotic sources. Processed ribosomes are severely depleted of initiation factors 2a-2° and endogenous messenger R N A activity, but will participate in translation with high efficiency when supplemented with these substances.Thus, the procedures which we have described are useful for preparing in vitro amino acid incorporation systems which can be used as sensitive assays for messenger RNA, tRNA, elongation factors, initiation factors, 2"-2e and other components of the protein biosynthetic apparatus which are usually associated with ribosomes prepared by other techniques. Acknowledgments We thank Ms. Marcia Witte for her excellent technical assistance and Demler Farms for the donation of adult Shaver Starcross Leghorn chickens.
[38]
The Characterization of Ribosomal Subunits Eukaryotes by Ultracentrifugal Techniques. Criteria of Purity
of
B y MARY G. HAMILTON
The problem of establishing homogeneity for a complicated structure like a ribosome is not easily solved, Perhaps such a concept should be irrevelant. The subunits of eukaryotic cytoplasmic ribosomes, in their "active" state, seem to behave more like supramolecular complexes than simple ribonucleoproteins.' Moreover, if heterogeneity exists among the population of small subunits of Escherichia coli ribosomes,'-' it probably exists for eukaryotic ribosomes as well. Despite these reservations it is important to try to characterize the subunits as fully as possible. Fortunately, techniques are now available for the complete characterization of ribosomal subunits.:' One can separate subunits by zonal centrifugation," although pressure-induced dissociation M. G. Hamilton, A. Pavlovec, and M. L. Petermann, Biochemistry 10, 3424 (1971). " C. G. Kurland, Annu. Rev. Biochem. 41, 377 (1972). ~M. G. Hamilton and M. E. Ruth, Biochemistry 8, 851 (1969). 'P. S. Sypherd and J. Wireman, this volume [35]; C. C. Sherton et al., this volume [36].
388
RIBOSOME STRUCTURE AND FUNCTION
~v 2
o O
v v v v Z
F~
Z
rnB
[38]
[38]
RIBOSOMAL SUBUNITS OF EUKARYOTES
389
may sometimes complicate the separation. 4~' The subunits can be fixed with formaldehyde to prevent degradation. This is useful in general but essential for the buoyant density measurements which can be used for an estimate of protein content as well as purity. The ultraviolet absorption optical system of the analytical ultracentrifuge permits measurements at very low concentrations. With only a few A260 units one can measure sedimentation coefficients and buoyant densities, and if the solutions are monodisperse, the molecular weight can be measured directly by equilibrium centrifugation, one of the few absolute methods. The table reproduces the results of such measurements on the subunits of rat liver ribosomes. Tashiro and Yphantis ~ and Hill et al. ~' have also used analytical ultracentrifugation to measure molecular weights of ribosomes and subunits. Criteria of P u r i t y : Sedimentation Analysis and B u o y a n t Density M e a s u r e m e n t s Although monodispersity of size is measurable by sedimentation analysis, 7 with ribosomal subunits a single sedimenting boundary may represent more than one species. Ribosomal subunits can both open up to sediment more slowly or dimerize to sediment faster. By some natural perverseness the opened large forms of rat liver ribosomes 8 sediment at almost exactly the same rate as active small subunits and, v i c e versa, dimerized active small ones sediment at almost exactly the same rate as large ones?' One simple test for such cross-contamination is to examine the sample after E D T A treatment. TM This converts the active forms to inactive forms of lower sedimentation (s) rates but has no effect on the "opened" forms. Examples of these cases are illustrated in Figs. 1 and 2 for fractions taken from zonal separations of ribosomes dissociated by the urea method of Petermann and Pavlovec. ~ In Fig. 1 small subunit dimers cosedimenting with the large subunits (upper left) are revealed by E D T A treatment as a 30 S boundary (lower left). In Fig. 2, an opened 41 S form of the large subunit cosedimenting with the small subunits (upper left) is unchanged by E D T A treatment (lower left), but the active 40 S small subunits now sediment at 28 S. Because the buoyant density in CsCI depends on the protein content 4,, R. Baierlein and A. A. Infante, this volume [33]. 5 y. Tashiro and D. A. Yphantis, J. Mol. Biol. 11, 174 (1965). "W. E. Hill, G. P. Rossetti, and K. E. Van Holde, J. Mol. Biol. 44, 263 (1969). 7R. Trautman, S. P. Spragg, and H. B. Halsall, Anal. Biochem. 28, 396 (1969). M. L. Petermann, A. Pavlovec, and M. G. Hamilton, Biochemistry 11, 3925 (1972). "M. G. Hamilton, unpublished observations, 1971. ~°M. L. Petermann, this series, Vol. 20, p. 429. 11M. L. Petermann and A. Pavlovec, Biochemistry 10, 2770 (1971).
390
RIBOSOME STRUCTURE AND FUNCTION
[38]
"~.7 S 15-
1.58 A
162
__.../299S
~
/
9.4S
FIG. 1. Large-subunit fraction from a zonal separation of rat liver ribosomes dissociated by treatment with 2 M urea. Comparison by sedimentation velocity (left side) and isodensity equilibrium centrifugation (right side) of samples fixed with H C H O before (upper row) and after (lower row) E D T A treatment. The patterns were obtained by densitometry of U V films.
152 1.53cj/ml
J
1.64
~40.5S
1.54
J'~" 28.4S > FIG. 2. Small subunit fraction from a zonal separation of rat liver ribosomes dissociated by urea. Upper patterns, control. Lower patterns, after E D T A treatment. See legend of Fig. 1. The patterns on the left were obtained by the photoelectric scanner, and the right-hand ones were obtained by densitometry of U V films.
[38]
RIBOSOMAL SUBUNITS OF EUKARYOTES
391
of the subunit and the active forms differ in protein content (see the table), another criterion of homogeneity is available. This analysis reveals crosscontamination of subunits even more dramatically than does the sedimentation pattern. Figure 1 shows a large subunit fraction examined in this way. While the sedimentation pattern shows mainly a single boundary (upper left), the isodensity pattern (upper right) shows three bands which correspond to small subunits, large subunits, and an intermediate band, which may represent residual ribosomes. Again, E D T A treatment (lower right) corroborates the existence of a mixture. The small subunit does not lose protein after E D T A treatment unless it is physically separated from the reaction mixture. ~' When fixed immediately after the addition of EDTA, it retains its low buoyant density. This is a convenience in this assay since it remains distinguishable from the large subunit, as is shown in Figs. 1 and 2, lower right panels. Technical Details The procedures for the main variations of analytical ultracentrifugation have been fully described in this series 1~ and elsewhere. 13 Chervenka" gives a simplified and practical guide. The calculations, with statistical treatment of the data, have been programmed for a desk-top computer? '~ We use Dr. Trautman's programs for the Olivetti Underwood Programma 101 Desk-Top Computer. Velocity sedimentation, in a density gradient with suitable markers of known s rates, and isodensity equilibrium centrifugation can be performed in preparative ultracentrifuges. Molecular weight measurements by classical equilibrium centrifugation, however, require an analytical ultracentrifuge. A minimum of 2 A~60 units, at a concentration of 0.6-0.8 A260 units/ml, is required for the measurements. Naturally, one would like more material for replicate analyses. Preparation o] Sample. Fixation with formaldehyde, which is necessary for the buoyant density measurement, is also useful in preserving the structure for the lengthy measurements. Buffers such as triethanolamine-HC1 (TEA-HC1), which lack primary amino groups, should be used rather than Tris. HC1. Fractions from the sucrose gradient separation are dialyzed overnight against 2 M formaldehyde, 10 mM TEA.HC1, pH 7.4, 0.2 mM 1, H. K. Schachman, this series, Vol. 4, p. 32; J. Vinograd, Vol. 6, p. 854. 13R. Trautman and M. G. Hamilton, in "Principles and Techniques in Plant Virology" (C. I. Kado and H. O. Agrawal, eds.), p. 491. Van Nostrand-Reinhold, Princeton, New Jersey, 1972. 1, C. H. Chervenka, "A Manual of Methods for the Analytical Ultracentrifuge." Spinco Division of Beckman Instruments, Inc., Palo Alto, California, 1969. 15R. Trautman, Ann. N.Y. Acad. Sci. 164, 52 (1969).
392
RIBOSOME STRUCTURE AND FUNCTION
[38]
MgCI2, and for 1 or 2 days against at least two lots of 0.1 M HCHO, 30 mM KCI, 10 mM TEA-HC1, pH 7.4, 0.2 mM MgC12 (FTKM), a suitable solvent for all the centrifugation studies. If necessary, the sample can be concentrated to 0.6-0.8 A260 units/ml by vacuum dialysis in a SchleicherSchuell collodion bag. EDTA Test for Cross-Contamination o[ Zonal Fractions. An excess of EDTA, 0.1 or 0.2 volumes of 0.1 M Na._, EDTA (depending on the MgC1._, content of the separation buffer), which has been adjusted to pH 7 with NaOH, is added to a 2 A_~60unit aliquot. After a few minutes the sample is dialyzed overnight against buffered 2 M HCHO without MgCle, but switched to FTKM for the final dialysis. Sedimentation Velocity. These runs should be performed first because the samples can be recovered after thorough mixing of the cell contents. The ultraviolet absorption optical system should be used preferably with the photoelectric scanner optics, but the film system can also be used. Multicell runs save time and improve accuracy by eliminating the temperature variable when comparisons of s rates are required. The FTKM buffer has a density of 1.0028 g/ml and a relative viscosity of 1.024. For this solvent and a partial specific volume, ~, of 0.63, the "salt correction" for calculating12,1' sedimentation in water, after the temperature correction to 20 ° has been made, is 1.03. Isodensity Equilibrium Centrilugation. These measurements have been described, a6 We now add 0.1 volume of 0.1 M NaH2PO4, 0.1 M Na2H PO4 to ensure a constant pH near 7.5.17 Also, the higher speeds available in a titanium rotor, 48,000 or 52,000 rpm, are advantageous in increasing the gradients and thus displaying more species. When small subunits are present, an initial density of 1.58 g/ml is useful. Classical Equilibrium Centrilugation. The requirements for monodispersity and lack of cross-contamination must be stressed. Since the high speed method is must be used (the present-day centrifuges are reputed to be unreliable at the lowest available speeds), complications due to the presence of heterogeneity should be avoided. The principles and practice of molecular weight measurements have been concisely described by Van Holde? 9 The photoelectric scanner optics must be used for UV measurements. The runs are made in the cold in the An-J rotor in multichannel cells as equipped with sapphire windows. No oil base is used. The solvent side ~6M. ~TH. D. 19K. of
G. Hamilton, this series, Vol. 20, p. 512. A. Wood, Virology 43, 511 (1971). A. Yphantis, Biochemistry 3, 297 (1964). E. Van Holde, "Sedimentation Equilibrium." Fractions I, p. 1. Spinco Division Beckman Instruments, Inc., Palo Alto, California, 1969.
[38]
RIBOSOMAL SUBUNITS OF EUKARYOTES
393
holds 0.13 ml of dialyzate and the sample side 0.10 ml of a solution of about 0.6 A._,6,, units/ml. Nine samples can be run at one time, and conveniently over the weekend; this is sufficient time to reach "practical" equilibrium, i.e., no change in the pattern with time. Figure 3 provides a guide for the selection of speed for the equilibrium run. This has been calculated from an equation given by Yphantis '~ for the desired concentration distribution. Reestablishing equilibrium at several higher speeds, e.g., 20% and 40% higher, improves precision by providing additional data. This usually takes an additional 24 hours for each new speed, and by clearing the meniscus area also provides baselines that can be used for the lower speeds. Establishment of the correct baseline is a problem with the high speed method. The solution columns can be scanned at 265 nm and 280 nm at the slowest scan speed to magnify the x dimension, x is converted to radius, r, in the usual way."-"" The y dimension is converted to absorbancy, c, by means of the calibration "steps" which accompany each scan. TM Since the absorbance at 280 nm is about half that at 260 nm, the 280 nm readings provide an optical dilution for the region near the base of the solution column. The data should be plotted on graph paper as log c vs. r-' and inspected for linearity. Frequently, points near the top of the solution column show a distinct curvature. This may indicate a poor choice of baseline, although heterogeneity can give a positive curvature and nonideality a negative one to the line. Readings less than 0.1 A unit probably should be discarded. Although the deficiencies of the log c vs. r ~ plot
15,000
-
I0,000c:::, 7,0005,000a:: 4,0000 5,000O3
2,000 -
1,000
0.1
012 0.[3 014015 017 I'.0 ]15 210 3'.0 41.050 7'0 MOL. WT x 10-6
FIG. 3. Graph for selection of speed for high speed equilibrium centrifugation of a ribonucleoprotein with ~ = 0.63, in a buffer with a density of 1.002 g / m l in a 3 mm column at 5 °. Adapted from K. E. Van Holde, "Sedimentation Equilibrium." Fractions 1, p. 1. Spinco Division, Beckman Instruments, Inc., Palo Alto, California, 1969.
394
RIBOSOME STRUCTURE AND FUNCTION
[38]
as an index of homogeneity have been noted, '-'° it is the simplest method to apply to scanner data. In the equation 4.606RT d(log c) (1 -- ~p)o~2 dfr 2)
Mw-
Mw is the weight-average molecular weight; R, the gas constant (8.313 X 10 r ergs deg-1 mol-0; T, the absolute temperature; p, the solution density (the solutions are so dilute that the solvent density of 1.002 g/ml may be used); ~, the partial specific volumeZl; % the angular velocity (2 rr rpm/60); and [d(log c)]/d(r'-') is the slope of the line obtained from the plot of log c vs. r -°. We have found ~2 that replicate measurements are essential for precision with the UV optical system (see also the table). When more material is available, the molecular weight measurements should be made with the interference optical system. This requires about 0.1 ml of a solution containing 4-6 A._,60units/ml. Plate reading is laborious (unless automated)~2% but more detailed and precise calculations of the molecular weight distribution can be madeY 3 Double exposures with schlieren optics, -~4 after masking of the interference image, provide additional data ( d c / d r ) for a plot of
the slope of which is proportional to M:, the Z-average molecular weight in the equation
Mz =
(1
-- ~p)o~2-
d(r ~)
Identity of Mw and M: is an indication of homogeneity. C o m m e n t s on O t h e r M e t h o d s
Obviously, with the chemical composition from the buoyant densityTM and the size of the RNA molecule, one can estimate the molecular weight of a ribosomal subunit. The molecular weight of RNA can be measured ~°H. Fujita and J. W. Williams, J. Phys. Chem. 70, 309 (1966). 21The partial specific volume can be estimated from the composition of the particle. In the expression, ~ = 0RNA( % R N A / 1 0 0 ) + ~protei,~ (%protein/100), ~a~.~ is 0.53 (measured pycnometrically for N a R N A ) and O~,~ot~,, is 0.74 (calculated from the amino acid composition). For rat liver ribosomal subunits this value coincides with the reciprocal of the buoyant density in CsC1, but for other ribosomes it may not. :ZM. G. Hamilton, Biochim. Biophys. Acta 134, 473 (1967). 2:~ Photographic labeling of fringes as suggested by S. J. Edelstein and G. H. Ellis [Analytical Biochemistry 43, 89 (1971)] helps in plate alignment on the microcomparator. :3D. C. Teller, T. A. Horbett, E. G. Richards, and H. K. Schachman, Ann. N.Y. Acad. Sci. 164, 66 (1969). 2~C. H. Chervenka, Anal. Chem. 38, 356 (1966).
[39]
HYDROGEN-TRITIUMEXCHANGE STUDIES ON RIBOSOMES
395
by sedimentation equilibrium,'-"-' but this requires purified RNA. A simple treatment with the detergent sodium dodecyl sulfate releases the R N A for examination by electrophoretic analysis in polyacrylamide gels or sedimentation analysis. Both methods can reveal the cross-contamination of large and small subunits discussed above, but obtaining the molecular weight by these methods may be a problem. The gel technique depends on calibration with molecules of known size and conformation and may be in serious error/'~ Estimating molecular weight from sedimentation velocity studies is also dependent on calibration curves or assumptions about shape and hydration. Sedimentation in formaldehyde is not the answer for eukaryotic ribosomal RNA's,',='" although it seems to work for bacterial ones and viral RNA's/G,'-'z A new electrophoretic technique using a nonaqueous solvent may prove useful. =~ ~-~I. B. Dawid and J. W. Chase, J. Mol. Biol. 63, 217 (1972). :" M. L. Fenwick, Biochem. J. 107, 851 (1968). "~H. Boedtker, J. Mol. Biol. 35, 61 (1968). ~ D. Z. Staynov, J. C. Pinder, and W. B. Gratzer, Nature (London) 235, 108 (1972).
[39]
Hydrogen-Tritium
Exchange
Studies on Ribosomes 1
B y MELVIN V. SIMPSON
The method of hydrogen-tritium exchange ( H X ) has been used in the study of some of the conformational properties of ribosomes/-s The method is very suitable for detecting changes in ribosomal conformation, particularly those which occur while the ribosome is functioning2 -7 Reviews on the theoretical aspects as well as the experimental aspects of H X have appeared 9,1° as well as a detailed description and discussion by 1Much of the research upon which this article is based was supported by Grants Nos. GB 5597 and GB 8375 from the National Science Foundation. -"L. A. Page, S. W. Englander, and M. V. Simpson, Biochemistry 6, 968 (1967). 3M. I. Sherman and M. V. Simpson, Cold Spring Harbor Syrup. Quant. Biol. 34, 220 (1969). ' M. I. Sherman, D. Chuang, and M. V. Simpson, Cold Spring Harbor Syrup. Quant. Biol. 34, 109 (1969). ~M. I. Sherman and M. V. Simpson, Proc. Nat. Acad. Sci. U.S. 64, 1388 (1969). '~D. Chuang, H. A. Silberstein, and M. V. Simpson, Arch. Biochem. Biophys. 144, 778 (1971). 7D. Chuang and M. V. Simpson, Proc. Nat. Acad. Sci. U,S. 68, 1474 (1971). M. I. Sherman, Eur. J. Biochem. 25, 291 (1972). ~A. Hvidt and S. O. Nielson, Advan. Protein Chem. 21, 287 (1966). :0 S. W. Englander, in "Poly-a-Amino Acids: Protein Models for Conformational Studies" (G. Fasman, ed.), p. 339. Dekker, New York, 1967.