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Electron microscopy of ribonucleoprotein particles from E. Coli
J.
]}I ol.
Biol. (1959) 1, 329-332
Electron Microscopy of Ribonucleoprotein Particles from E. Coli C. E.
HALL AND H. S. SLAYTER
Department of Biology, .1}l assachusetts Institute of T echnology, Cambridge, Massachusetts, U.S.A . (Received 8 August 1959) Four kinds of ribonucleoprotein particles fr om E. coli charact eri zed by the sedimentation constant s 30, 50, 70 and 1008 were obser ve d in shadow cast sp ecimens and mea sured. The 30 and 508 particles are ap pare n t ly produced by a splitting of the 708, while the 1008 material consists of dimers of the 70S which form at high concentrations of Mg++. As dried and prepared for electron microscopy, 308 particles are asymmetric, representable by a prolate ellipsoid 95 X 170 A. The 508 p articles are more nearly spherical and r epresented by an obiate ellipsoid 170 X 140 A. Volumes calculated fr om these dimensions are in excell ent agreement with th ose deduced from physical chem ica l data. 708 particles exh ib it an undefined su bst r uc t ure and are not closely representable as ellip soids, but the diameter and h eight are est imated to be approxima tely 200 and 170 A respectively. The results in general eon firm and supplement the physical che m ica l st udies of Ti ssieres a nd co- wo rkers on these same m ateri al s.
1. Introduction
Tissieres & Watson (1958) and 'I'issieres, Watson, Schlessinger & H ollingworth (1959) have described the isolation of ribonucleoprotein (RNP) particles from E. coli and the separat ion of this material into four components characterized by sedimentation constants of 30,50,70 and 100S. We report here electron microscopic observations of samples of these materials made concurrently with the studies which they have described.
2. Materials and Methods Samples suspended in magnesium acetate prepared by Tissieres et al, (1959) were transported packed in ice immed ia tely after isolation a n d prepared for electron microscopy by a spray drop m ethod (Hall, 1956) within a few hours. Polystyrene latex spheres with an average diameter of 880 A were added to the preparations to provide an internal stan dard for measuring the sh adow -t o-h eigh t ratio. Specimen s were shadow cast with platinum at an angle of about 5 : 1. Initial experiments indicated that the particles were r elatively unstable in time and that their st ab ilit y was also a function of Mg++ concentration (T'iseiercs & Watson, 1958). The r esults to be de s cribed were obtained from materials sus pen ded in various concentrati ons of m agnesium acetate, t h ou gh we generally u se volat ile salts in this spray m ethod. Althou gh magnesium acetate is non-volatile, its presence did not ca us e noticeable deter ioration of the images in the concen t rat ions u sed, which were 5 X 10- 3 M an d less.
3. Observations and Results (a) jl1orphology of the RNP particles Electron micrographs of the four components are shown in Plate 1. The 30 S comp onent is the smallest and consists t o a large exte nt of particles which are rodshaped as indicated by the arrow in Plate I(a). The 50S component shown in Plate 329
C. E. HALL AND H. S. SLAYTER
330
I(b) is more nearly spherical in appearance. The 70S specimens always contained an appreciable number of particles similar in size to those in the 30 and 508 materials as well as some larger particles indicated by the arrows in Plate I(c). These latter particles show a complexity of structure as though they were made up of subunits, but the appearance is so variable that a unique morphology has not been determined. The 1008 preparation contained a large number of diploid particles as indicated by the arrow in Plate I(d). In many instances these particles show one or more small granules lodged in between the two larger symmetrical units. This appearance is to be seen in an exaggerated fashion in the particle in the lower right hand corner of Plate I(d) where we see a diploid stretched out, showing two large particles separated by two smaller subunits, all in a line. (b) Measurements of 30,50 and 708 preparations In the case of the 50 and 708 particles two measurements were made from electron micrographic prints (at about 100,000 x): (a) widths perpendicular to the shadow 40 30 S
20
N
50S 40
r
20
0f-----,,..---,--F=--'r---'-.....-='-r--.,--,--+--,.-.dJL>.r"-"'L 70 S
20
100
200 WIDTH IN
~
300
400
0
FIG. 1. Histograms showing widths and heights of platinum-shadowed ribonucleoprotein particles from E. coli, characterized by their sedimentation constants. In the case of the 308 particles the "width" refers to the long axis and for the other two the width is measured perpendicular to the shadow direction. The widths include a cap of metal which is estimated to exaggerate this dimension by about 60 A.
direction and (b) shadow lengths from which particle heights can be calculated. In the case of the 308 particles which are somewhat rod-shaped as indicated by the arrow in Plate I(a) the longest dimension was measured rather than the width perpendicular to the shadow direction. Results of these two measurements for about 100 particles in each case are shown in Fig. 1 for 30, 50 and 708 preparations. The apparent width is about the same for the 30 and 50S particles. In the 70S preparation
PLATE 1. Ribonucleoprotein particles from E. coli. Magnification 100,000 X. (a) RXP particles from a 30 s preparation. The arrow indicates a typical asymmetrical particle. (b) RNP particles from a 50 s preparation. (c) RNP particles from a 70 s preparation. These specimens also show 30 and 50 s particles. The 70 s particles, such as are indicated by arrows, show a substructure as though they consisted of two or more granules. (d) RNP particles from a 100 s preparation. The 100 s particles are believed to be dirners of the 70 s. [To face page 330
R IB O N UC LEO P R O TE I N PAR T IC L ES
331
there is also a large number with about the same width as the ot hers, but there is a significant number wit h a greater widt h. The height of t he 30 S p articles is significantly smaller than the height of the 50 S. The 70 S shows a relatively wide distribution of heights, some about t he same as t he 30 and 50 S a nd some whi ch are significantly higher. The 70 S preparation apparently contains breakdown products resulting fr om preparative procedures for electron mi croscopy (EM). The distribution of measurements can be account ed for by considering it to consist of a mixture of 30 Sand 50 S p ar ti cles and a third larger component, presumably 70 S . The experiment sho wn in Fig. 1 was rep eat ed on a second set of materials wit h practicall y identical results. From measurem ent s of a number of spherica l molecules of known molecular weight we have found that t he measured shadow len gths are a good measure of t he true particle h eigh ts and that apparent widths are t oo great in our m ethod by ab out 60 A du e t o t he cap of sha dowi ng metal. With t hese considerations in mind t he analysis of the results yields t wo figures, a h eigh t and a width (corrected for metal), whi ch figures we assume t o be the maj or axes of equivalent ellipsoids for the purpose of calculating molecular volumes for comp arison with the data of Tissieres et al. (1959). For t he 70 S particles we h ave selected t h e dimensions of the larger particles in t he field, ignoring the smaller par ti cles of about the dimensions of the 30 and 50 S preparati ons. Figures for t he 30 and 50 S particles are averages a nd in clude more particles than are represent ed in Fi g. 1. A sum mary of average dimensions is shown in Ta ble 1 T ABLE
Sedimentation constant
Mg++
EM
EM
D im ensions A
M.W. X 10- 6
Ti ssieres et al. M.W. X 10-6
P r olate 30S
2 X
lO- 4M
95 X 170
0'76
0' 70
2'0
I' SO
3' 4
2'63
Ob la te 50S
2' 5 X
lO-4It!
170 X 140
Oblate 70S
1O- 3 l\1
200 X 170
where the partial specific volume 0·64 is use d t o convert calcula te d molecular volume to molecular weigh t. Since the vo lume is qui t e sensitive to linear dimensions the agreement between EM and physical chemical methods is very good for the 30 and 50 S particles though the EM value is 10% high in bot h cases. In the case of the 70 S preparation t he EM value is too high in comparison wit h the physical chemical dat a and also 20% greater t han t he su m of t he 30 a nd 50 S weights from electron micro scopy. We take t his to mea n t hat the 70 S p ar t icles are not acc urately represent a ble as a solid ellipsoid . I t is not clea r in det ail from t he micrographs how t he 30 a nd 50S p arti cles migh t be pu t toget her to form a 70 S. They are probably not t o be thought of as rigid structures that retain exactly t he sa me shape wh en t hey separate. (c) Comparison oj 70 and 100 S preparations
Two sa mples containing fairly high Mg++ and characterized by the sedimentation constants 70 Sand 100 S respectively were examined. Both of these preparations showed t wo peaks in t he ultracent rifu ge and ar e cha racte rized by t he const ant for t he larger peak (Tissieres & Watson, 1958). Micrographs of bot h of t hese preparations
C. E. HALL AND H. S. SLAYTER
332
showed a relatively large number of double particles as indicated by the arrow in Plate I(d) as well as single particles such as one sees in 70 8 preparations. Fi elds were selecte d at random and counts were made of the number of single and double particles present in both pr eparations. The relative amount of material present in the two form s is shown in Table 2 for each preparation, on the assumption that a double TABLE 2
Material
" 70 S " " I OOS "
~Ig ++
0'002 M 0'00 5 111
N cou n te d
406 229
Frac t ion of ma terial t in: single particl es double particles
65 % 39 %
35 % 61 %
tAssuming that the mass of a d ouble particle is twice that of a singl e.
particle has twice the mass of a single. (This is not strictly accurate since there are some single particles smaller than those identified as 70 8 .) The increase in double particles at the higher Mg++ concentration leaves little doubt that the 1008 peak in the centrifuge is to be identified with the double particles. Particle volumes for the dim ers estimated from shadow lengths and widths are abo ut t wice the volum e of the 70 8 particles.
4. Conclusions In general, the observations confirm the results of Tissieres et al. (1959) on the exist ence and weights of th e four components of RNP designated 30,50,70 and 100 8 . In addition, we observe aspects of the particle morphology which supplements t he evidence from physical chemical methods. The 308 particles have a high degr ee of asymmetry (axial ratio ab out 2 : 1.) The 508 particles are more nearly sph erieal.Tl'he slight oblateness observed may be partly caused by a flattening against the support ing surface.) The 708 particles exhibit a substruct ure which is not sufficiently well-defined to show clearly how the 30 and 508 fragments are related when combined in the 708 . Due to irregularities in shape , the 708 particles are not closely representable by a solid ellipsoid with axes corresponding to the major dimensions. The 100 S particles appear clearly to consist of two 70 S particles combined. This investigation was supported by a research grant C-2171 (C6 & 7) from the National Cancer Institute of the Nation al Institutes of Health, United States Public Health Service.
REFERENCES H all , C. E. (1956). J . B iophys . B iochem , Oytol. 2, 625. 'I'issieres, H. & Watson , J. D. (1958). N ature, 182, 778. Tissieres, A., Watson , J . D. , Sch lessinger , D. & Hollingworth, B . R. (1959). J. Mo l. B iol. 1, 221.
]}I ol.
Biol. (1959) 1, 329-332
Electron Microscopy of Ribonucleoprotein Particles from E. Coli C. E.
HALL AND H. S. SLAYTER
Department of Biology, .1}l assachusetts Institute of T echnology, Cambridge, Massachusetts, U.S.A . (Received 8 August 1959) Four kinds of ribonucleoprotein particles fr om E. coli charact eri zed by the sedimentation constant s 30, 50, 70 and 1008 were obser ve d in shadow cast sp ecimens and mea sured. The 30 and 508 particles are ap pare n t ly produced by a splitting of the 708, while the 1008 material consists of dimers of the 70S which form at high concentrations of Mg++. As dried and prepared for electron microscopy, 308 particles are asymmetric, representable by a prolate ellipsoid 95 X 170 A. The 508 p articles are more nearly spherical and r epresented by an obiate ellipsoid 170 X 140 A. Volumes calculated fr om these dimensions are in excell ent agreement with th ose deduced from physical chem ica l data. 708 particles exh ib it an undefined su bst r uc t ure and are not closely representable as ellip soids, but the diameter and h eight are est imated to be approxima tely 200 and 170 A respectively. The results in general eon firm and supplement the physical che m ica l st udies of Ti ssieres a nd co- wo rkers on these same m ateri al s.
1. Introduction
Tissieres & Watson (1958) and 'I'issieres, Watson, Schlessinger & H ollingworth (1959) have described the isolation of ribonucleoprotein (RNP) particles from E. coli and the separat ion of this material into four components characterized by sedimentation constants of 30,50,70 and 100S. We report here electron microscopic observations of samples of these materials made concurrently with the studies which they have described.
2. Materials and Methods Samples suspended in magnesium acetate prepared by Tissieres et al, (1959) were transported packed in ice immed ia tely after isolation a n d prepared for electron microscopy by a spray drop m ethod (Hall, 1956) within a few hours. Polystyrene latex spheres with an average diameter of 880 A were added to the preparations to provide an internal stan dard for measuring the sh adow -t o-h eigh t ratio. Specimen s were shadow cast with platinum at an angle of about 5 : 1. Initial experiments indicated that the particles were r elatively unstable in time and that their st ab ilit y was also a function of Mg++ concentration (T'iseiercs & Watson, 1958). The r esults to be de s cribed were obtained from materials sus pen ded in various concentrati ons of m agnesium acetate, t h ou gh we generally u se volat ile salts in this spray m ethod. Althou gh magnesium acetate is non-volatile, its presence did not ca us e noticeable deter ioration of the images in the concen t rat ions u sed, which were 5 X 10- 3 M an d less.
3. Observations and Results (a) jl1orphology of the RNP particles Electron micrographs of the four components are shown in Plate 1. The 30 S comp onent is the smallest and consists t o a large exte nt of particles which are rodshaped as indicated by the arrow in Plate I(a). The 50S component shown in Plate 329
C. E. HALL AND H. S. SLAYTER
330
I(b) is more nearly spherical in appearance. The 70S specimens always contained an appreciable number of particles similar in size to those in the 30 and 508 materials as well as some larger particles indicated by the arrows in Plate I(c). These latter particles show a complexity of structure as though they were made up of subunits, but the appearance is so variable that a unique morphology has not been determined. The 1008 preparation contained a large number of diploid particles as indicated by the arrow in Plate I(d). In many instances these particles show one or more small granules lodged in between the two larger symmetrical units. This appearance is to be seen in an exaggerated fashion in the particle in the lower right hand corner of Plate I(d) where we see a diploid stretched out, showing two large particles separated by two smaller subunits, all in a line. (b) Measurements of 30,50 and 708 preparations In the case of the 50 and 708 particles two measurements were made from electron micrographic prints (at about 100,000 x): (a) widths perpendicular to the shadow 40 30 S
20
N
50S 40
r
20
0f-----,,..---,--F=--'r---'-.....-='-r--.,--,--+--,.-.dJL>.r"-"'L 70 S
20
100
200 WIDTH IN
~
300
400
0
FIG. 1. Histograms showing widths and heights of platinum-shadowed ribonucleoprotein particles from E. coli, characterized by their sedimentation constants. In the case of the 308 particles the "width" refers to the long axis and for the other two the width is measured perpendicular to the shadow direction. The widths include a cap of metal which is estimated to exaggerate this dimension by about 60 A.
direction and (b) shadow lengths from which particle heights can be calculated. In the case of the 308 particles which are somewhat rod-shaped as indicated by the arrow in Plate I(a) the longest dimension was measured rather than the width perpendicular to the shadow direction. Results of these two measurements for about 100 particles in each case are shown in Fig. 1 for 30, 50 and 708 preparations. The apparent width is about the same for the 30 and 50S particles. In the 70S preparation
PLATE 1. Ribonucleoprotein particles from E. coli. Magnification 100,000 X. (a) RXP particles from a 30 s preparation. The arrow indicates a typical asymmetrical particle. (b) RNP particles from a 50 s preparation. (c) RNP particles from a 70 s preparation. These specimens also show 30 and 50 s particles. The 70 s particles, such as are indicated by arrows, show a substructure as though they consisted of two or more granules. (d) RNP particles from a 100 s preparation. The 100 s particles are believed to be dirners of the 70 s. [To face page 330
R IB O N UC LEO P R O TE I N PAR T IC L ES
331
there is also a large number with about the same width as the ot hers, but there is a significant number wit h a greater widt h. The height of t he 30 S p articles is significantly smaller than the height of the 50 S. The 70 S shows a relatively wide distribution of heights, some about t he same as t he 30 and 50 S a nd some whi ch are significantly higher. The 70 S preparation apparently contains breakdown products resulting fr om preparative procedures for electron mi croscopy (EM). The distribution of measurements can be account ed for by considering it to consist of a mixture of 30 Sand 50 S p ar ti cles and a third larger component, presumably 70 S . The experiment sho wn in Fig. 1 was rep eat ed on a second set of materials wit h practicall y identical results. From measurem ent s of a number of spherica l molecules of known molecular weight we have found that t he measured shadow len gths are a good measure of t he true particle h eigh ts and that apparent widths are t oo great in our m ethod by ab out 60 A du e t o t he cap of sha dowi ng metal. With t hese considerations in mind t he analysis of the results yields t wo figures, a h eigh t and a width (corrected for metal), whi ch figures we assume t o be the maj or axes of equivalent ellipsoids for the purpose of calculating molecular volumes for comp arison with the data of Tissieres et al. (1959). For t he 70 S particles we h ave selected t h e dimensions of the larger particles in t he field, ignoring the smaller par ti cles of about the dimensions of the 30 and 50 S preparati ons. Figures for t he 30 and 50 S particles are averages a nd in clude more particles than are represent ed in Fi g. 1. A sum mary of average dimensions is shown in Ta ble 1 T ABLE
Sedimentation constant
Mg++
EM
EM
D im ensions A
M.W. X 10- 6
Ti ssieres et al. M.W. X 10-6
P r olate 30S
2 X
lO- 4M
95 X 170
0'76
0' 70
2'0
I' SO
3' 4
2'63
Ob la te 50S
2' 5 X
lO-4It!
170 X 140
Oblate 70S
1O- 3 l\1
200 X 170
where the partial specific volume 0·64 is use d t o convert calcula te d molecular volume to molecular weigh t. Since the vo lume is qui t e sensitive to linear dimensions the agreement between EM and physical chemical methods is very good for the 30 and 50 S particles though the EM value is 10% high in bot h cases. In the case of the 70 S preparation t he EM value is too high in comparison wit h the physical chemical dat a and also 20% greater t han t he su m of t he 30 a nd 50 S weights from electron micro scopy. We take t his to mea n t hat the 70 S p ar t icles are not acc urately represent a ble as a solid ellipsoid . I t is not clea r in det ail from t he micrographs how t he 30 a nd 50S p arti cles migh t be pu t toget her to form a 70 S. They are probably not t o be thought of as rigid structures that retain exactly t he sa me shape wh en t hey separate. (c) Comparison oj 70 and 100 S preparations
Two sa mples containing fairly high Mg++ and characterized by the sedimentation constants 70 Sand 100 S respectively were examined. Both of these preparations showed t wo peaks in t he ultracent rifu ge and ar e cha racte rized by t he const ant for t he larger peak (Tissieres & Watson, 1958). Micrographs of bot h of t hese preparations
C. E. HALL AND H. S. SLAYTER
332
showed a relatively large number of double particles as indicated by the arrow in Plate I(d) as well as single particles such as one sees in 70 8 preparations. Fi elds were selecte d at random and counts were made of the number of single and double particles present in both pr eparations. The relative amount of material present in the two form s is shown in Table 2 for each preparation, on the assumption that a double TABLE 2
Material
" 70 S " " I OOS "
~Ig ++
0'002 M 0'00 5 111
N cou n te d
406 229
Frac t ion of ma terial t in: single particl es double particles
65 % 39 %
35 % 61 %
tAssuming that the mass of a d ouble particle is twice that of a singl e.
particle has twice the mass of a single. (This is not strictly accurate since there are some single particles smaller than those identified as 70 8 .) The increase in double particles at the higher Mg++ concentration leaves little doubt that the 1008 peak in the centrifuge is to be identified with the double particles. Particle volumes for the dim ers estimated from shadow lengths and widths are abo ut t wice the volum e of the 70 8 particles.
4. Conclusions In general, the observations confirm the results of Tissieres et al. (1959) on the exist ence and weights of th e four components of RNP designated 30,50,70 and 100 8 . In addition, we observe aspects of the particle morphology which supplements t he evidence from physical chemical methods. The 308 particles have a high degr ee of asymmetry (axial ratio ab out 2 : 1.) The 508 particles are more nearly sph erieal.Tl'he slight oblateness observed may be partly caused by a flattening against the support ing surface.) The 708 particles exhibit a substruct ure which is not sufficiently well-defined to show clearly how the 30 and 508 fragments are related when combined in the 708 . Due to irregularities in shape , the 708 particles are not closely representable by a solid ellipsoid with axes corresponding to the major dimensions. The 100 S particles appear clearly to consist of two 70 S particles combined. This investigation was supported by a research grant C-2171 (C6 & 7) from the National Cancer Institute of the Nation al Institutes of Health, United States Public Health Service.
REFERENCES H all , C. E. (1956). J . B iophys . B iochem , Oytol. 2, 625. 'I'issieres, H. & Watson , J. D. (1958). N ature, 182, 778. Tissieres, A., Watson , J . D. , Sch lessinger , D. & Hollingworth, B . R. (1959). J. Mo l. B iol. 1, 221.