Biochimica et Biophysica Acta, l 130 (1992) 289-296 © 1992 El~vier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00
289
BBAEXP 92365
Characteristic electron microscopical projections of the small ribosomal subunit from Thermomyceslanuginosus George Harauz and Derrick Flannigan Department of Molecular Biology and Genetics, Unh'ersity of Guelph, Guelph (Canada) (Received 21 August 1991) (Revised manuscript received 18 December 1991)
Key words: Ribosome; Thermophile; Fungus; Electron microscopy;Image analysis
Multivariate statistical analysis and hierarchical ascendant classification techniques have been used to sort electron images of small ribosomal subunits from the thermophilic fungus Thennomycesianuginosus into their characteristic views. Three predominant modes of adsorption to the support were elucidated: right-lateral, left-lateral and asymmetric, showing reproducible detail approaching 1.8 nm resolution. The projections of the fungal complexes appeared almost identical to those of HeLa cells, rat liver and rabbit reticulocytes studied previously in this manner. This result contra~s with the greater variation in fine structural features observed between ribosomal subunits from different prokaryotic species. 2 Introduction
The computational analysis of electron micrographs of ribosomes and ribosomal subunits via multivariate statistical techniques (correspondence analysis) has been invaluable in elucidating fine structural details in a quantitative and objective manner (reviewed in Refs. 1-3). Ribosomal structures from diverse species of prokaryotes, and especially Escherichia colt, have been extensively studied in attempts to answer questions about the structure of this macromolecular complex and to use such structural information in phylogenetic analyses (e.g., Refs. 4,5). Eukaryotic ribosomes have received considerably less attention than those from prokaryotes, and to date few comparative studies employing image processing have been performed [3,6-9]. In a previous paper [8], we described the computational analysis of electron micrographs of whole cytoplasmic ribosomes from Thermomyces lanuginosus, sometimes referred to as Humicola lanuginosa, a mildly thermophilic fungus which grows optimally at a temperature of about 55°C [10]. Three predominant projection views of the monosome were constructed, called overlap, non-overlap, and top, showing reproducible detail approaching 1.8 nm resolution. No major morphological differences could be detected between the
Correspondence: G. Harauz, Department of Molecular Biology and Genetics, University of Guelph, Guelph, Ontario, Canada, NIG 2Wl.
fungal and eubacterial (E. colt) ribosomes at the resolution achieved, despite the evolutionary gap between these two organisms. In this paper, we describe the results of electron image analysis of the small ribosomal subunit of Th. lanuginosus and compare the characteristic views obtained with those of subunits from other eukaryotes [4,6,7,9, l l, 12]. Materials and Methods
Th. lanuginosus was grown in starch broth at 55°C with agitation for 60-72 h and the mycelium harvested by filtration. All subsequent steps were at 4°C, using autoclaved buffers and glassware. The mycelium (2.5-3 g) was ground with alumina powder (5 g) for 10-15 min using a mortar and pestle, after which four volumes of homogenisation buffer (20 mM Tris-HCI (pH 7.5), 5 mM I~.g(CH3COO)2, 10 mM KCI, 6 mM 2-mercaptoethanol, 25 units/ml RNAguard (Pharmacia)) were added and mixed. The sample was centrifuged at 8000 rpm (5000 x g ) in a Beckman JA20 rotor for 30 min. The supernatant was removed and centrifuged again at 18000 rpm (25300 x g ) for 45 min in a JA20 rotor. This supernatant was layered over a cushion of 30% sucrose in homogenisation buffer, in a volume ratio of 2:1, and centrifuged at 35 000 rpm (93 000 × g) for 20 h in a Beckman 70 Ti rotor. The pellet, which contained ribosomes, was rinsed three times with sterile double distilled water. To dissociate ribosomes into subunits, the pellet was resuspended in 400/zl of dissociation buffer (50 mM
290 Tris,HC! (pH 7.5), 12 mM Mg(CH3COO) 2, 0.75 M KCI, 4 mM 2-mercaptoethanol, and I 0 0 / t g / m l heparin added fresh). This suspension was layered over a linear sucrose gradient (8-40% in dissociation buffer). After spinning for 5 h at 38000 rpm (190000×g, ~ c k m a n SW 41 rotor) the gradient was fractionated from using a Haake:Buchler Densi, Flow IIC fractionator (Canlab)and the absorbance at 254 nm of the eluate was continually monitored using a dual path monitor UV-2 (Pharmacia). Fractions enriched in small subunits were pooled. Purified small ribosomal subunits were prepared for electron microscopy by adsorption to a single layer of carbon film and negatively contrasted with 2% uranyl acetate. Electron micrographs were taken on a JEOL J EM-100CX at an instrument magnification of 50000 x and an accelerating voltage of 80 kV. The grids were consistently inserted into the microscope with the specimen side oriented away from the electron source. Each specimen area was not preilluminated prior to being micrographed, to minimisc the total electron dose. Six electron micrographs were digitised using an Optronics rotating drum densitometer and a scanning step size of 25 /~m, corresponding to 0.5 nm at the object level. During digitisation, the emulsion of the negative was kept down facing ir*.o the drum. Digitised images were transferred via magnetic tape to an IRIS 3120 workstation (Silicon Graphics, Mountain View, CA). All distinct complexes that were not in close contact with others were selected interactively using the IRIS monitor and mouse controlled cursor. A total of 1040 particles was selected, i.e., subimages of size 90 x 90 pixels and containing a single ribosomal subunit within them were extracted from the larger micrographs. Subsequent 'single particle' data analysis steps have been described in detail in previously published papers and reviews [2,5,8] and are only summarised here. The 1040 ribosomal subunit images were pretreated by band-pass filtering, surrounded by a circular mask, floated within this mask to a zero average density and given a normalised variance. Two images were selected after an exhaustive visual survey to use as references for alignment - these represented the so-called leftand right-lateral views of the subunit [6,7]. The entire data set was aligned with respect to each of these references in turn, with the final alignment parameters for each image chosen on the basis of highest correlation coefficient between the aligned and reference images. This step is called multi-reference alignment (MRA). Then multivariate statistical analysis (MSA), hie~rchical ascendant classification (HAC), and summing of class members were performed [2,5,8]. The two best class averages representing the left- and rightlateral views were used as references for another MRA,
following which MSA/HAC were performed again. These references were better than the original ones since sources of random noise (e.g., stain variations) were averaged out to become less significant compared to the common signal, viz., the projected structure of the subunit in a specific orientation. The second round of MSA/HAC yielded a third dominant projection, the asymmetric one. AnOther three class averages representing the three different views were used as references for a final round of MRA and MSA/HAC. A total of 12 eigenimages was used in the HAC step to yield the class averages reported here. (Strictly speaking, the correct word in the realm of abstract mathematics is 'eigenvectors', our preference is for 'eigenimages' which has more precise meaning in the context of electron microscopy.) In other words, the first 12 factorial coordinates were used to calculate interimage distances for HbC. In order to refine the MSA/HAC results further, all images belonging to the best class averages representing the three different projections were extracted from the larger data set and analysed separately. The total sum of each subset was used to create a binary mask within which to perform a new MSA analysis. The purpose of the MSA mask was to remove from consideration those regions of the image that did not belong to the complex under study, i.e., the background carbon support. Since the size and nature of the mask can potentially influence the results, it was appropriate to analyse more homogeneous groupings of different projections separately in an attempt to distinguish further subtler morphological features. Results and Discussion
The computational analysis of electron micrographs of 1040 small ribosomal subunits from Th. lanuginosus enabled the grouping together of those subunits lying in the same orientation on the support film. Images within each group could then be averaged together to give a picture in which noise, due to factors such as variable stain distribution and an uneven carbon background, was reduced with respect to the common structural signal. The 'best' classes resulting from the HAC were selected on the bases of having at least 10 members and of having structural congruity with other class averages, i.e., in not being atypical (Fig. 1). Other class averages were of poor quality in that they comprised mainly poorly aligned images, or images inadequately represented by the factorial coordinates defined by the MSA, and were not considered in subsequent analyses. In this population of images, the ribosomal subunits exhibited three preferred modes of orientation: leftlateral (Fig. 1, averages 1-11), right-lateral (Fig. 1, averages 12-16) and asymmetric (Fig. 1, averages 1720). The names 'left-lateral' and 'right-lateral' were
291
Fig. 1. Class averages representing reproducible modes of adsorption to the carbon support of the small ribosomal subunit of Th. hmuginosus. The numbers of images contained within e:leh class are 21 (average No. 1), 20 (No. 2), 37 (No. 3), 17 (No. 4), 19 (No. 5), 17 (No. 6), 19 (No. 7), 14 (No. 8), 31 (No. 9), 35 (No. 10), 21 (No. 1!), 26 (No. 12), 23 (No. 13), 21 (No. 14), 39 (No. 15). 20 (No. 16), 26 (No. 17), 33 (No. 18), 26 (No. 19), 12 (No. 20), representing 477 particles, or 47% of the total population of 1040.
chosen on the basis of the direction that the upper 'beak' pointed in. Within the starting set of 1040 images, 251 fell into good left-lateral classes, 129 into right-lateral classes and 97 into asymmetric classes. Two of the best class averages (in terms of internal homogeneity, i.e., a relatively low intraclass variance) of each of these projections are shown in Fig. 2, with contour lines superimposed. For comparison, a gallery of aligned subunits lying in the left-lateral orientation is shown in Fig. 3. In these figures, it should be noted that intermediate spatial frequency detail has been emphasised by the initial band-pass filtering operation. The major structural landmarks in the left- and right-lateral views of the small ribosomal subunit of Th. lamlginosus are the prominent beak, the two 'feet' at the bottom, and two lobes of stain exclusion at the back (called the 'back lobes' in Ref. 9 and the 'platform' in Ref. 4). These lateral views are not exact mirror versions of each other because of the orienta. tion dependent interaction with negative stain. This is especially ,evident in comparing the back lobes, in the asymmetric view, the head is strongly stain excluding and the asymmetry is due to the small arm protruding towards the viewer's right. A stained region down the
Fig. 2. Selected class averages derived from the final MSA and classificationof the aligned data set, grouped according to view: left-lateral (I). right-lateral (r), asymmetric(a). The scale bar represents l0 nm.
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Fig, 3, Selection of aligned Th, ImeuSinosi~s small ribosomal subunits lying in the left-lateral orientation•
middle of the particle may represent either a surface trough or a stretch of increased positive staining. The bead:body di,~,ision is roughly one third to two thirds. The visual quality of the class averages (Figs. 1, 2) is much better than of the original images (Fig. 3) and finer morphological detail can be elucidated with greater confidence. An objective measure of the resolution of detail in these images is obtained by the
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we used before [5,8]. The reproducible spatial resolutions of the left-lateral, right-lateral and asymmetric views of Fig. 2 are approx. 2.2 nm, 1.8 nm and 2.5 rim, respectively. These resolution limits are appropriate for images of negatively stained preparations of this type, and serve primarily as a rough measure of the degree of betterment in image quality achieved by averaging• In contrast to the Fourier Ring Correlation [5,8], the information measure described in Ref. 13 provides an estimate of reproducible spatial resolution within a collection of aligned images, rather than be-
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Fig. 5.'Plot of positions of 1040 ribosomal subunit images in a two-dimensional space represented by factorial coordinates I and 2 (panel 1). Plots of positions of images belonging to left-lateral classes (panel 2), right-lateral classes (panel 3) and asymmetrical classes (panel 4). When the left-lateral, right-lateral and asym.metric clusters have been subtracted away, the remaining points at the top and center left of this map represent those images that did not fall into the selected best classes (Fig. 1), due primarily to orientational misalignment.
293 tween two distinct images whose equivalence must be assumed. A total of 27 eigenimages representing 68.1% of the total interimage variance were calculated, but only the first 12 were used (equally weighted) in the HAC step to yield the class averages reported here. These eigenimages are shown in Fig. 4. The distributions of the entire set of images and the three characteristic views in a two-dimensional space represented by the first two principal features are shown in Fig. 5. The first two eigenimages appear to define the distinction amongst the three predominant characteristic views. Eigenimages 3 to 5 emphasise the differences in peripheral staining. The higher order ones describe subtler variations in both external and internal stain distribution. The physical reasons for interimage variability are factors such as the variability of local staining, conformational or structural heterogeneity, or orientational differences ('rocking'). Referring back to the reproducible spatial resolutions derived for each projection average, and all other factors being equal, it may be conjectured that the asymmetric and left-lateral views exhibit a greater range of rocking than the right-lateral one. This idea has been proposed for small ribosomal subunits of E. colt [14], although this phenomenon was not reproduced in other studies by different groups [15]. The MSA was performed on image pixels contained within a contour derived from the total sum of 1024 particles in the whole set. The extent of this mask is seen in Fig. 4 which shows the eigenimages. The MSA/HAC performed within this mask allowed not only the distinction of the predominant characteristic projections, but also variations of appearance within each of these three major groupings. Returning to Fig. 1, there are obvious differences in all views in the amount of penetration of negative stain into peripheral cavities, especially the one separating the head from the body of the subunit. Within both lateral groupings, the back lobes vary in relative degree of stain exclusion. In order to inquire further into the subtle variability of morphological features (Fig. 1), the individual images representing each distinct projection were assembled into files containing 251, 129 and 85 particles (images of the rotated asymmetric class, No. 20 in Fig. 1, were not used). The total sums of each characteristic view are shown in Fig. 6. Contours around each particle in the total average were used to define new masks within which to perform M S A / H A C separately. The first 12 eigenimages yielded by these new MSAs are shown in Fig. 7, and were used as before in the HAC procedure to generate the class averages shown in Fig. 8. As would be expected from MSAs of more homogeneous data sets, the first factorial coordinates account for greater proportions (32.9%, 34.5%, 44.2%) of the total interimage variapce than that of the whole data
Fig. 6. Total averages of the left-lateral, right-lateral and asymmetric views. An interactive display program was used to fi~rm contours around each average, thereby defining masks for MSA/HAC of the more homogeneous subsets.
Fig. 7. (a) Left-lateral eigenimages 1--12, representing the basis vectors corresponding to factorial coordinates i-12. and acc,.)unting for 32.9%, 3,5%, 2.2%, 2.0%, 1.8¢~..... and 1.3~ of the interimage variance in the data set (cumulative tot~l is 52.6'~). (b) Righ~i-lateral eigenimages !-12, accounting for 34.5%, 3.(1%, 2.4%, 2.2%, 2.1% ..... and 1.5% of the interimage variance in Lhe data set (cumulative total is 56.4%). (c) Asymmetric eigenimages 1-12. accounling for 44.2"~.~. 3.0%, 2.8%, 2.7%, 2.2% ..... and 1.5~,, of the interimage variance: in the data set (cumulative total is 67.6%).
Fig. 8. Class averages derived from individual analyses of subsets of the three predominant projection views. The number of images contained within each class is 62 (No. I), 42 (No. 2~ 44 (No. 3}. 38 (No. 4), 35 (No. 5), 30 (No. 61, 38 (No. 7). 40 (No. 8), 2(I (No, 9). 31 (No. 101, 30 (No. IlL 55 (Nc~. 12).
294 set (25.5%) [16]. The new eigenimages (Fig. 7) can be interpreted as before, although the vicissitude of peripheral local staining appears to be represented primarily by the 2nd eigenimage of each set. The cutoff criteria of the final HACs were chosen to increase the average numbers of members per class, and thereby ~tentially improve the signal-to-noise ratio further. A comparison of the class averages of Fig. 8 with those of Fig. 1 shows a good correspondence in that the same major morphological features are apparent, in this study, then, results obtained by the MSA/HAC were not overly sensitive to changes in size and shape of the masks imposed on the images for these procedures. The variability of morphological features in the two-dimensional projections is thus real in this data set, reflecting most probably the phenomena of inconstant staining and rocking as ascertained in Ref, 7. The averaging of more (beyond a certain point) images within a class does not yield appreciably better results, as would be expected from previous work [6]. Our work bears direct comparison with electron image analyses of small ribosomal subunits from HeLa cells [6,7] and rabbit reticulocytes [9]. When correspondence analysis was first introduced to electron microscopy of biological macromolecules, Frank et al. [6,7] determined that the peripheral stain density around each subunit was the major source of heterogeneity between images of different particles. These workers then constructed separate averages of both the left- and right-lateral views of the eukaryotic small subunit, and were first to note the different 'backward slantingness' of the platform in these projections. The m~or structural features (beak. feet and back lobes) were shown to be significant, and their peculiarity to eukaryotes was later used for phylogenetic classifications of organisms based on micrographs of ribosomal structures [4], it was of interest to us here that our averages of the left and right-lateral projections of the Th, Immginosus small ribosomal subunit appeared almost identical to Frank et al.'s corresponding ones from HeLa cells. More recently, Verschoor et al. [9] computed a three-dimensional reconstruction of the eukaryotic small ribosomal subunit from rabbit reticulocytes. It was seen from the reconstruction that the right-lateral view corresponded to a viewing angle of about 180°, and the left-lateral view corresponded to a viewing angle of - 2 0 °, explaining in part fl~rther the inexact symmetry, Although this group did not separately extract and analyse the asymmetric view that we found here. it appears to correspond to a viewing angle of roughly 2800-300 °, based on their demonstration of a broad, hollowed-out trough region on the interface side of the complex. The asymmetric view described here agrees with visual studies of its appearance in micrographs of subunits from rat liver and rabbit
ret~cuiocytes [11] and wheat germ [12], as well as having been previously characterised for prokaryotes [14,15]. Bota lateral views predominated in the Th. lanuginosus preparation, consistent with these previous studies, although no 'quasi-symmetric' projection class [11,12,14,15] was elucidated here. In addition to investigating the appearance in electron micrographs of the eukaryotic ribosomal small subunit, Frank et al. [7] used correspondence analysis to inquire into the nature of negative staining and its effects on the appearance of two-dimensional electron microscopical projections of ribosomal subunits. In reference [7], a single carbon preparation was used. For purposes of three-dimensional reconstruction, pceparations of ribosomes sandwiched between two carbon layers (the 'sandwich" technique) were better [9]. (The early literature on electron microscopy of ribosomes is not clear on this point, as sometimes both approaches appeared to be used interchangeably [11].) Further and most recent discussion on this subject is found in Frank [1.7], and the main tenets submitted are relevant here. Frank's group identified the variability of the negative stain distribution on the periphery of the particles, and noted that these particles exhibited small ranges of preferred orientations. They concluded from their own and other investigations that the double carbon approach had advantages of ensuring complete enibedment of the specimen so as to be able to consider it a real projection for reconstruction, and that significant protection against radiation damage was conferred. Thus, this preparation approach has become the most common in the field. In this paper as well as in previous work [2,8], our own preference has been for single carbon preparations for a number of reaso~s. First of all, the single carbon preparatio~ is far easier and consequently appears to be more reproducible in our hands. Secondiy, we have noted il~traspecies variability in the appearance of archaebacterial prokaryotic large ribosoma~ subunits even i~J double carbon preparations [Z]. Thirdly, from a Lacge population of 1956 large ribosomal subunits from E. coli imaged on a single carbon layer, we elucidated rarely occurring views which could he interpreted as pr,~jections :lifferent from those represented by the most commonly occurring motifs [2]. These experiences suggest to us that the variability observed here in fine morphological features within subunits of Th. lanuginosus, prepared using the single carbon approach, would not necessarily be eradicated by the double carbon technique, and that there may be advantages to the former. Finally, our concurrent work on electron spectroscopic imaging of the ribosomal phosphorus distribution in situ requires the use of unstained specimens and an exceedingly thin carbon support (ideally of the order of 2-3 nm) (cf., Ref. 18). Thus, the consistent use of single carbon preparations
295 within our laboratory appears to be appropriate in order to reconcile results obtained by the new imaging modes, it is noteworthy that the averages of large numbers of particles exhibiting the lateral views (Fig. 6) show greater similarity between these two projections. We regard this phenomenon as analogous to the observation that the two lateral views of HeLa small subunits grew increasingly alike as the depth of staining increased [7]. These reflections affirm that the issue of degree of staining should always be kept in mind, but suggest that the choice of single vs. double carbon preparations does not affect the conclusions reached here. This question becomes less serious especially when quantitative electron image analysis is exploited to select the best class averages (Fig. 2). In a review entitled 'Evolutionary conservation of structure and function of high molecular weight ribosomal RNA' [19], Rau6 et al. distinguish the major classes of ribosomes to be those from eubacteria, archaebacteria, the cytoplasm of eukaryotes, plastids of plants and algae, mitochondria of plants and fungi, and mitochondria of mammals. Even within these larger groupings, there is a considerable variation in the size and number of ribosomal RNA and protein constituents. At present, our knowledge of the comparative structures of cytoplasmic eukaryotic ribosomes comprises mainly sequence data. The known cytoplasmic eukaryotic small ribosomal subunit RNAs are generally about 1800 nucleotides in length, although extremely large or small ones exist in some organisms. The comparison of many such sequences enables the identification of conserved helical (base-pairing) regions. The proposed secondary structures of small subunit ribosomal RNAs from most organisms are in many ways comparable to that of the E. coil paradigm [19], in that there is a universally conserved structural core which presumably is minimally necessary for proper ribosome functioning. However, there are still many differences in secondary structure of this ribosomal RNA species between prokaryotes and eukaryotes, and also among eukaryotes, that arise within these conserved helices as well as within the less-conserved variable regions. In contrast to the large numbers of ribosomal RNA sequences that have been and continue to be accumulated, there is a paucity of ribosomal protein sequences [20]. No set of cytoplasmic eukaryotic ribosomal proteins has yet been completely defined, although it is likely that there are specific ones unique to eukaryotes. At present, though, interspecies comparisons of ribosome structure are based primarily on ribosomal RNA data. The important question remains how differences in ribosomal RNA secondary structure, as well as diversity in protein composition, are reflected in the three-dimensional structures of these complexes. It has been noted previously that the small ribosomal subunits from many organisms exhibit a greater similarity
in many physical properties than the corresponding large subunits [4,21,22]. It was anticipated at the onset of our studies of Th. lanuginosus ribosomes and subunits that structural differences would be revealed with the aid of electron image analysis. However, alternative approaches such as electron spectroscopic imaging [18,23] or scanning transmission electron microscopy [24] of the ribosomal RNA component selectively, and in situ, may be more efficacious in this regard. Th. lanuginosus excretes amylolytic (starch degrading) enzymes which are of interest for biotechnological applications because of their increased thermostability [25]. The small subunit ribosomal RNA components and large subunit ribosomal RNA genes of this organism have also been examined in the framework of larger investigations on eukaryotic RNA structure and processing [26-29] and RNA-protein interaction [31)1. The presence of conserved A and T rich segments within the generally stabler G and C rich nucleic acids has allowed the identification of domains functionally important in termination and maturation. The ribosomes of this fungus have not otherwise been studied. Because of their strongly conserved eukaryotic structural characteristics and increased thermostability, ribosomal subunits and proteins from Th. lam~ginosus may be attractive candidates for attempts at crystallisation and X-ray diffractometry [31], and thus deserve further investigation. However, standard techniques of electron microscopy alone are of diminished utility in defining characters for phylogenetic categorisation within the eukaryotic kingdom, because of the structural congruity of the small subunit to the resolutions achievable. In summary, we have constructed characteristic projection views of the small ribosomal subunit from the fungus Tit. latmginosus. The averages we obtained corresponded to and appeared ahnost identical to those derived from other eukaryotic species by similar computational approaches. Previous comparative studies of ribosomal subunits from diverse prokaryotic species showed considerable variation in fine structural detail. Our present results provide further evidence that small ribosomal subunit structure is strongly conserved within eukaryotes, despite even the temperature dependence of ribosome function within thermophiles (Ref. 32, cf. also Ref. 33). This knowledge is of significance in consideration of future studies of cukaryotic ribosomal structure and function, showing the latitude to which experimental results can be extrapolated to different species.
Acknowledgements This work was supported by grants from the Research Advisory Board of the University of Guelph. and the Natural Sciences and Engineering Research
296 Council of Canada to G.H. We are grateful to D) Patrick Whippey (University of Western Ontario) fo~ assistance with the densitometry. References I Frank, J,, Radermacher, M., Wagenknecht, T. and Verschoor, A. {1988) Methods Enzs'mol. 164, 3-35. 2 Harauz, G., Boekema, E. and Van Heel, M. (1988) Methods En~mol. 164, 35-49. 3 Frank, J., Verscboor, A., Radermacher, M. and Wagenknecht, T. 0990) in The Ribosome: Structure, Function, and Evolution, (Hill, W.E., Dahlberg, A., Garrett, R.A., Moore, P.B., Schle~inger, D. and Warner, J.R,, eds.), pp, 107-113, American Society for Microbiology, Washington, 4 Hcnde~on, E,, Oakcs, M,, Clark, M,W., Lake, J.A, Matheson, A,T, and gillig, W. (1984) Science 2~, 510-512. 5 Harauz, G,, St6ffler-Meilicke, M. and Van I1e¢1, M, (1987) J. Mol, Evol, 26, 347-357, 6 Frank, J,, Verschoor, A, and Boublik, M, (1981) Science 214, 1353-1355, 7 Frank, J,, Ve~choor, A. and Boublik, M, (19821J. Mol. Biol. 161, 107-137, 8 tlarauz, G. :rod Flannigan, D, (19901 Biochim, Biophys. Acta 1038, 260-267, 9 Verschoor, A,, Zhang, N..Y,, Wagenkneeht, T,, Obrig, T., Ruderreacher, M. aad Frank, J, (1989)J. Mol. Biol. 209, 115-126. I0 Domsch, K,H,, Gums, W. and Ander~n, T.-H. (1980) Compendium of Soil Fungi, Academic Press, New York. II lvanov, I,E, and Sabatini, D.R. (19811 J. UItrastruct. Res. 76, 263-276. 12 Monte,~ano, L. and Glitz, D.G. (19881 J, Biol. Chem, 263, 49324938. 13 Sass, J.P., Biildt, G., Beckmann, E., Zemlin, F., Van Heel, M,, Zeitler, E., Rosenbusch, J.P., Dorset, D.L. and Massalski, A. (1989) J. Mol. Biol. 209, 171-175. 14 Ve1~choor, A,, Frank, J., Radermacher, M., Wagenknecht, T. and l~.)ublik,M. (1984) J, Mol. Biol. 178, 677-698, 15 Van Heel, M, and St~.~fflcr-Meilicke.M, (1985) E M B O J. 4, 2389=2395, 16 Borland, L, and Van Hccl, M. (19)0~ J. Opt. Soc, America A 7, 601-610,
17 Frank, J. (1989) Electron Microsc. R~v. 2, 53-74. 18 Ottensmeyer, F.P. (19841 J, Ultrastruct.Res. 88, 121-134. 19 Rau6, H.A., Klootwijk, J. and Musters. W. (1988) Prog. Biophys. Mol. Biol.51, 77-129. 20 Wittmann-Liebold, B., K6pke, A.K.E., Arndt, E., Kr6mer, W., Hatakeyama, T. and Wittmann, H,-G. (19901 in The Ribosome: Structure, Function and Evolution, {Hill, W.E., Dahlberg, A., Garrett, R.A., Moore, P,B., Schlessinger,D. and Warner, J.R., eds.),pp. 598-616, American Society for Microbiology, Washington. 21 Cammarano, P., Romeo, A., Gentile, M., Felsani, A, and Gualerzi, C. (1972) Biochim. Biophys. Acta 281,597-624. 22 Boublik, M,, and Hellmann, W. and Jenkins, F, {1982) Proc, 10th Intern. Cong. Electron Microsc. 3, 95-96. 23 Korn, A.P., Spitnik-El~n, P., Elson, D, and Ottensmeyer, F.P. (19831 Eur. J. Cell Biol. 31,334-340. 24 Boublik, M,, Tumminia, S.J., Hellmann, W., Zhang, Q., Hainfield,J.F. and Wall, J.S. (1991) in Proc. 49th Ann. Meet. Elect. Microsc. Soc, Am, (Bailey, G.W. and Hall, E.L., eds.),pp. 262263, San Francisco Press, San Francisco. 25 Jenssen, B., Olsen, J. and Allermann, K. (1988) Can. J. Microbiol. 34, 218-223, 26 Wildeman, A.G. and Nazar, R.N. (1981) J. Biol. Chem. 256, 5675-5682. 27 Nazar, R,N., Wong, W,M. and Abrahamson, J,L.A. (1987) J. Biol. Chem, 262, 7523-7527. 28 Walker, K., Wong, W.M. and Nazar, R.N. {199{|) Mol. Call. Biol. Ill,377-381, 29 Sutherland, L.A., Wong, W.M. and Nazar, R.N. (1989) Nucleic Acids Res. 17, 10504. 30 Nazar, R.N. and Wildeman, A.G. (1983) Nucleic Acids Res. II, 3155-3168. 31 Yonath, A., Bennett, W., Weinstein, S., and Wittmann, H.-G. (1990) in The Ribosome: S~ructure, Function, and Evolution, (Hill, W.E., Dahlberg, A., Garrelt, R.A., Moore, P.B., Schlessinger, D. and Warner, J.R,, eds.L pp. 134-147, American Society for Microbiology, Washington. 32 Miller, H.M. and Shepherd. M.G. (1973~ Can. J. Microbiol. 19, 761-763. 33 Boublik. M. and Ramagopal, S. (1980) Mol. Gen. Genet. 179, 483-488.
289
BBAEXP 92365
Characteristic electron microscopical projections of the small ribosomal subunit from Thermomyceslanuginosus George Harauz and Derrick Flannigan Department of Molecular Biology and Genetics, Unh'ersity of Guelph, Guelph (Canada) (Received 21 August 1991) (Revised manuscript received 18 December 1991)
Key words: Ribosome; Thermophile; Fungus; Electron microscopy;Image analysis
Multivariate statistical analysis and hierarchical ascendant classification techniques have been used to sort electron images of small ribosomal subunits from the thermophilic fungus Thennomycesianuginosus into their characteristic views. Three predominant modes of adsorption to the support were elucidated: right-lateral, left-lateral and asymmetric, showing reproducible detail approaching 1.8 nm resolution. The projections of the fungal complexes appeared almost identical to those of HeLa cells, rat liver and rabbit reticulocytes studied previously in this manner. This result contra~s with the greater variation in fine structural features observed between ribosomal subunits from different prokaryotic species. 2 Introduction
The computational analysis of electron micrographs of ribosomes and ribosomal subunits via multivariate statistical techniques (correspondence analysis) has been invaluable in elucidating fine structural details in a quantitative and objective manner (reviewed in Refs. 1-3). Ribosomal structures from diverse species of prokaryotes, and especially Escherichia colt, have been extensively studied in attempts to answer questions about the structure of this macromolecular complex and to use such structural information in phylogenetic analyses (e.g., Refs. 4,5). Eukaryotic ribosomes have received considerably less attention than those from prokaryotes, and to date few comparative studies employing image processing have been performed [3,6-9]. In a previous paper [8], we described the computational analysis of electron micrographs of whole cytoplasmic ribosomes from Thermomyces lanuginosus, sometimes referred to as Humicola lanuginosa, a mildly thermophilic fungus which grows optimally at a temperature of about 55°C [10]. Three predominant projection views of the monosome were constructed, called overlap, non-overlap, and top, showing reproducible detail approaching 1.8 nm resolution. No major morphological differences could be detected between the
Correspondence: G. Harauz, Department of Molecular Biology and Genetics, University of Guelph, Guelph, Ontario, Canada, NIG 2Wl.
fungal and eubacterial (E. colt) ribosomes at the resolution achieved, despite the evolutionary gap between these two organisms. In this paper, we describe the results of electron image analysis of the small ribosomal subunit of Th. lanuginosus and compare the characteristic views obtained with those of subunits from other eukaryotes [4,6,7,9, l l, 12]. Materials and Methods
Th. lanuginosus was grown in starch broth at 55°C with agitation for 60-72 h and the mycelium harvested by filtration. All subsequent steps were at 4°C, using autoclaved buffers and glassware. The mycelium (2.5-3 g) was ground with alumina powder (5 g) for 10-15 min using a mortar and pestle, after which four volumes of homogenisation buffer (20 mM Tris-HCI (pH 7.5), 5 mM I~.g(CH3COO)2, 10 mM KCI, 6 mM 2-mercaptoethanol, 25 units/ml RNAguard (Pharmacia)) were added and mixed. The sample was centrifuged at 8000 rpm (5000 x g ) in a Beckman JA20 rotor for 30 min. The supernatant was removed and centrifuged again at 18000 rpm (25300 x g ) for 45 min in a JA20 rotor. This supernatant was layered over a cushion of 30% sucrose in homogenisation buffer, in a volume ratio of 2:1, and centrifuged at 35 000 rpm (93 000 × g) for 20 h in a Beckman 70 Ti rotor. The pellet, which contained ribosomes, was rinsed three times with sterile double distilled water. To dissociate ribosomes into subunits, the pellet was resuspended in 400/zl of dissociation buffer (50 mM
290 Tris,HC! (pH 7.5), 12 mM Mg(CH3COO) 2, 0.75 M KCI, 4 mM 2-mercaptoethanol, and I 0 0 / t g / m l heparin added fresh). This suspension was layered over a linear sucrose gradient (8-40% in dissociation buffer). After spinning for 5 h at 38000 rpm (190000×g, ~ c k m a n SW 41 rotor) the gradient was fractionated from using a Haake:Buchler Densi, Flow IIC fractionator (Canlab)and the absorbance at 254 nm of the eluate was continually monitored using a dual path monitor UV-2 (Pharmacia). Fractions enriched in small subunits were pooled. Purified small ribosomal subunits were prepared for electron microscopy by adsorption to a single layer of carbon film and negatively contrasted with 2% uranyl acetate. Electron micrographs were taken on a JEOL J EM-100CX at an instrument magnification of 50000 x and an accelerating voltage of 80 kV. The grids were consistently inserted into the microscope with the specimen side oriented away from the electron source. Each specimen area was not preilluminated prior to being micrographed, to minimisc the total electron dose. Six electron micrographs were digitised using an Optronics rotating drum densitometer and a scanning step size of 25 /~m, corresponding to 0.5 nm at the object level. During digitisation, the emulsion of the negative was kept down facing ir*.o the drum. Digitised images were transferred via magnetic tape to an IRIS 3120 workstation (Silicon Graphics, Mountain View, CA). All distinct complexes that were not in close contact with others were selected interactively using the IRIS monitor and mouse controlled cursor. A total of 1040 particles was selected, i.e., subimages of size 90 x 90 pixels and containing a single ribosomal subunit within them were extracted from the larger micrographs. Subsequent 'single particle' data analysis steps have been described in detail in previously published papers and reviews [2,5,8] and are only summarised here. The 1040 ribosomal subunit images were pretreated by band-pass filtering, surrounded by a circular mask, floated within this mask to a zero average density and given a normalised variance. Two images were selected after an exhaustive visual survey to use as references for alignment - these represented the so-called leftand right-lateral views of the subunit [6,7]. The entire data set was aligned with respect to each of these references in turn, with the final alignment parameters for each image chosen on the basis of highest correlation coefficient between the aligned and reference images. This step is called multi-reference alignment (MRA). Then multivariate statistical analysis (MSA), hie~rchical ascendant classification (HAC), and summing of class members were performed [2,5,8]. The two best class averages representing the left- and rightlateral views were used as references for another MRA,
following which MSA/HAC were performed again. These references were better than the original ones since sources of random noise (e.g., stain variations) were averaged out to become less significant compared to the common signal, viz., the projected structure of the subunit in a specific orientation. The second round of MSA/HAC yielded a third dominant projection, the asymmetric one. AnOther three class averages representing the three different views were used as references for a final round of MRA and MSA/HAC. A total of 12 eigenimages was used in the HAC step to yield the class averages reported here. (Strictly speaking, the correct word in the realm of abstract mathematics is 'eigenvectors', our preference is for 'eigenimages' which has more precise meaning in the context of electron microscopy.) In other words, the first 12 factorial coordinates were used to calculate interimage distances for HbC. In order to refine the MSA/HAC results further, all images belonging to the best class averages representing the three different projections were extracted from the larger data set and analysed separately. The total sum of each subset was used to create a binary mask within which to perform a new MSA analysis. The purpose of the MSA mask was to remove from consideration those regions of the image that did not belong to the complex under study, i.e., the background carbon support. Since the size and nature of the mask can potentially influence the results, it was appropriate to analyse more homogeneous groupings of different projections separately in an attempt to distinguish further subtler morphological features. Results and Discussion
The computational analysis of electron micrographs of 1040 small ribosomal subunits from Th. lanuginosus enabled the grouping together of those subunits lying in the same orientation on the support film. Images within each group could then be averaged together to give a picture in which noise, due to factors such as variable stain distribution and an uneven carbon background, was reduced with respect to the common structural signal. The 'best' classes resulting from the HAC were selected on the bases of having at least 10 members and of having structural congruity with other class averages, i.e., in not being atypical (Fig. 1). Other class averages were of poor quality in that they comprised mainly poorly aligned images, or images inadequately represented by the factorial coordinates defined by the MSA, and were not considered in subsequent analyses. In this population of images, the ribosomal subunits exhibited three preferred modes of orientation: leftlateral (Fig. 1, averages 1-11), right-lateral (Fig. 1, averages 12-16) and asymmetric (Fig. 1, averages 1720). The names 'left-lateral' and 'right-lateral' were
291
Fig. 1. Class averages representing reproducible modes of adsorption to the carbon support of the small ribosomal subunit of Th. hmuginosus. The numbers of images contained within e:leh class are 21 (average No. 1), 20 (No. 2), 37 (No. 3), 17 (No. 4), 19 (No. 5), 17 (No. 6), 19 (No. 7), 14 (No. 8), 31 (No. 9), 35 (No. 10), 21 (No. 1!), 26 (No. 12), 23 (No. 13), 21 (No. 14), 39 (No. 15). 20 (No. 16), 26 (No. 17), 33 (No. 18), 26 (No. 19), 12 (No. 20), representing 477 particles, or 47% of the total population of 1040.
chosen on the basis of the direction that the upper 'beak' pointed in. Within the starting set of 1040 images, 251 fell into good left-lateral classes, 129 into right-lateral classes and 97 into asymmetric classes. Two of the best class averages (in terms of internal homogeneity, i.e., a relatively low intraclass variance) of each of these projections are shown in Fig. 2, with contour lines superimposed. For comparison, a gallery of aligned subunits lying in the left-lateral orientation is shown in Fig. 3. In these figures, it should be noted that intermediate spatial frequency detail has been emphasised by the initial band-pass filtering operation. The major structural landmarks in the left- and right-lateral views of the small ribosomal subunit of Th. lamlginosus are the prominent beak, the two 'feet' at the bottom, and two lobes of stain exclusion at the back (called the 'back lobes' in Ref. 9 and the 'platform' in Ref. 4). These lateral views are not exact mirror versions of each other because of the orienta. tion dependent interaction with negative stain. This is especially ,evident in comparing the back lobes, in the asymmetric view, the head is strongly stain excluding and the asymmetry is due to the small arm protruding towards the viewer's right. A stained region down the
Fig. 2. Selected class averages derived from the final MSA and classificationof the aligned data set, grouped according to view: left-lateral (I). right-lateral (r), asymmetric(a). The scale bar represents l0 nm.
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middle of the particle may represent either a surface trough or a stretch of increased positive staining. The bead:body di,~,ision is roughly one third to two thirds. The visual quality of the class averages (Figs. 1, 2) is much better than of the original images (Fig. 3) and finer morphological detail can be elucidated with greater confidence. An objective measure of the resolution of detail in these images is obtained by the
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we used before [5,8]. The reproducible spatial resolutions of the left-lateral, right-lateral and asymmetric views of Fig. 2 are approx. 2.2 nm, 1.8 nm and 2.5 rim, respectively. These resolution limits are appropriate for images of negatively stained preparations of this type, and serve primarily as a rough measure of the degree of betterment in image quality achieved by averaging• In contrast to the Fourier Ring Correlation [5,8], the information measure described in Ref. 13 provides an estimate of reproducible spatial resolution within a collection of aligned images, rather than be-
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Fig. 5.'Plot of positions of 1040 ribosomal subunit images in a two-dimensional space represented by factorial coordinates I and 2 (panel 1). Plots of positions of images belonging to left-lateral classes (panel 2), right-lateral classes (panel 3) and asymmetrical classes (panel 4). When the left-lateral, right-lateral and asym.metric clusters have been subtracted away, the remaining points at the top and center left of this map represent those images that did not fall into the selected best classes (Fig. 1), due primarily to orientational misalignment.
293 tween two distinct images whose equivalence must be assumed. A total of 27 eigenimages representing 68.1% of the total interimage variance were calculated, but only the first 12 were used (equally weighted) in the HAC step to yield the class averages reported here. These eigenimages are shown in Fig. 4. The distributions of the entire set of images and the three characteristic views in a two-dimensional space represented by the first two principal features are shown in Fig. 5. The first two eigenimages appear to define the distinction amongst the three predominant characteristic views. Eigenimages 3 to 5 emphasise the differences in peripheral staining. The higher order ones describe subtler variations in both external and internal stain distribution. The physical reasons for interimage variability are factors such as the variability of local staining, conformational or structural heterogeneity, or orientational differences ('rocking'). Referring back to the reproducible spatial resolutions derived for each projection average, and all other factors being equal, it may be conjectured that the asymmetric and left-lateral views exhibit a greater range of rocking than the right-lateral one. This idea has been proposed for small ribosomal subunits of E. colt [14], although this phenomenon was not reproduced in other studies by different groups [15]. The MSA was performed on image pixels contained within a contour derived from the total sum of 1024 particles in the whole set. The extent of this mask is seen in Fig. 4 which shows the eigenimages. The MSA/HAC performed within this mask allowed not only the distinction of the predominant characteristic projections, but also variations of appearance within each of these three major groupings. Returning to Fig. 1, there are obvious differences in all views in the amount of penetration of negative stain into peripheral cavities, especially the one separating the head from the body of the subunit. Within both lateral groupings, the back lobes vary in relative degree of stain exclusion. In order to inquire further into the subtle variability of morphological features (Fig. 1), the individual images representing each distinct projection were assembled into files containing 251, 129 and 85 particles (images of the rotated asymmetric class, No. 20 in Fig. 1, were not used). The total sums of each characteristic view are shown in Fig. 6. Contours around each particle in the total average were used to define new masks within which to perform M S A / H A C separately. The first 12 eigenimages yielded by these new MSAs are shown in Fig. 7, and were used as before in the HAC procedure to generate the class averages shown in Fig. 8. As would be expected from MSAs of more homogeneous data sets, the first factorial coordinates account for greater proportions (32.9%, 34.5%, 44.2%) of the total interimage variapce than that of the whole data
Fig. 6. Total averages of the left-lateral, right-lateral and asymmetric views. An interactive display program was used to fi~rm contours around each average, thereby defining masks for MSA/HAC of the more homogeneous subsets.
Fig. 7. (a) Left-lateral eigenimages 1--12, representing the basis vectors corresponding to factorial coordinates i-12. and acc,.)unting for 32.9%, 3,5%, 2.2%, 2.0%, 1.8¢~..... and 1.3~ of the interimage variance in the data set (cumulative tot~l is 52.6'~). (b) Righ~i-lateral eigenimages !-12, accounting for 34.5%, 3.(1%, 2.4%, 2.2%, 2.1% ..... and 1.5% of the interimage variance in Lhe data set (cumulative total is 56.4%). (c) Asymmetric eigenimages 1-12. accounling for 44.2"~.~. 3.0%, 2.8%, 2.7%, 2.2% ..... and 1.5~,, of the interimage variance: in the data set (cumulative total is 67.6%).
Fig. 8. Class averages derived from individual analyses of subsets of the three predominant projection views. The number of images contained within each class is 62 (No. I), 42 (No. 2~ 44 (No. 3}. 38 (No. 4), 35 (No. 5), 30 (No. 61, 38 (No. 7). 40 (No. 8), 2(I (No, 9). 31 (No. 101, 30 (No. IlL 55 (Nc~. 12).
294 set (25.5%) [16]. The new eigenimages (Fig. 7) can be interpreted as before, although the vicissitude of peripheral local staining appears to be represented primarily by the 2nd eigenimage of each set. The cutoff criteria of the final HACs were chosen to increase the average numbers of members per class, and thereby ~tentially improve the signal-to-noise ratio further. A comparison of the class averages of Fig. 8 with those of Fig. 1 shows a good correspondence in that the same major morphological features are apparent, in this study, then, results obtained by the MSA/HAC were not overly sensitive to changes in size and shape of the masks imposed on the images for these procedures. The variability of morphological features in the two-dimensional projections is thus real in this data set, reflecting most probably the phenomena of inconstant staining and rocking as ascertained in Ref, 7. The averaging of more (beyond a certain point) images within a class does not yield appreciably better results, as would be expected from previous work [6]. Our work bears direct comparison with electron image analyses of small ribosomal subunits from HeLa cells [6,7] and rabbit reticulocytes [9]. When correspondence analysis was first introduced to electron microscopy of biological macromolecules, Frank et al. [6,7] determined that the peripheral stain density around each subunit was the major source of heterogeneity between images of different particles. These workers then constructed separate averages of both the left- and right-lateral views of the eukaryotic small subunit, and were first to note the different 'backward slantingness' of the platform in these projections. The m~or structural features (beak. feet and back lobes) were shown to be significant, and their peculiarity to eukaryotes was later used for phylogenetic classifications of organisms based on micrographs of ribosomal structures [4], it was of interest to us here that our averages of the left and right-lateral projections of the Th, Immginosus small ribosomal subunit appeared almost identical to Frank et al.'s corresponding ones from HeLa cells. More recently, Verschoor et al. [9] computed a three-dimensional reconstruction of the eukaryotic small ribosomal subunit from rabbit reticulocytes. It was seen from the reconstruction that the right-lateral view corresponded to a viewing angle of about 180°, and the left-lateral view corresponded to a viewing angle of - 2 0 °, explaining in part fl~rther the inexact symmetry, Although this group did not separately extract and analyse the asymmetric view that we found here. it appears to correspond to a viewing angle of roughly 2800-300 °, based on their demonstration of a broad, hollowed-out trough region on the interface side of the complex. The asymmetric view described here agrees with visual studies of its appearance in micrographs of subunits from rat liver and rabbit
ret~cuiocytes [11] and wheat germ [12], as well as having been previously characterised for prokaryotes [14,15]. Bota lateral views predominated in the Th. lanuginosus preparation, consistent with these previous studies, although no 'quasi-symmetric' projection class [11,12,14,15] was elucidated here. In addition to investigating the appearance in electron micrographs of the eukaryotic ribosomal small subunit, Frank et al. [7] used correspondence analysis to inquire into the nature of negative staining and its effects on the appearance of two-dimensional electron microscopical projections of ribosomal subunits. In reference [7], a single carbon preparation was used. For purposes of three-dimensional reconstruction, pceparations of ribosomes sandwiched between two carbon layers (the 'sandwich" technique) were better [9]. (The early literature on electron microscopy of ribosomes is not clear on this point, as sometimes both approaches appeared to be used interchangeably [11].) Further and most recent discussion on this subject is found in Frank [1.7], and the main tenets submitted are relevant here. Frank's group identified the variability of the negative stain distribution on the periphery of the particles, and noted that these particles exhibited small ranges of preferred orientations. They concluded from their own and other investigations that the double carbon approach had advantages of ensuring complete enibedment of the specimen so as to be able to consider it a real projection for reconstruction, and that significant protection against radiation damage was conferred. Thus, this preparation approach has become the most common in the field. In this paper as well as in previous work [2,8], our own preference has been for single carbon preparations for a number of reaso~s. First of all, the single carbon preparatio~ is far easier and consequently appears to be more reproducible in our hands. Secondiy, we have noted il~traspecies variability in the appearance of archaebacterial prokaryotic large ribosoma~ subunits even i~J double carbon preparations [Z]. Thirdly, from a Lacge population of 1956 large ribosomal subunits from E. coli imaged on a single carbon layer, we elucidated rarely occurring views which could he interpreted as pr,~jections :lifferent from those represented by the most commonly occurring motifs [2]. These experiences suggest to us that the variability observed here in fine morphological features within subunits of Th. lanuginosus, prepared using the single carbon approach, would not necessarily be eradicated by the double carbon technique, and that there may be advantages to the former. Finally, our concurrent work on electron spectroscopic imaging of the ribosomal phosphorus distribution in situ requires the use of unstained specimens and an exceedingly thin carbon support (ideally of the order of 2-3 nm) (cf., Ref. 18). Thus, the consistent use of single carbon preparations
295 within our laboratory appears to be appropriate in order to reconcile results obtained by the new imaging modes, it is noteworthy that the averages of large numbers of particles exhibiting the lateral views (Fig. 6) show greater similarity between these two projections. We regard this phenomenon as analogous to the observation that the two lateral views of HeLa small subunits grew increasingly alike as the depth of staining increased [7]. These reflections affirm that the issue of degree of staining should always be kept in mind, but suggest that the choice of single vs. double carbon preparations does not affect the conclusions reached here. This question becomes less serious especially when quantitative electron image analysis is exploited to select the best class averages (Fig. 2). In a review entitled 'Evolutionary conservation of structure and function of high molecular weight ribosomal RNA' [19], Rau6 et al. distinguish the major classes of ribosomes to be those from eubacteria, archaebacteria, the cytoplasm of eukaryotes, plastids of plants and algae, mitochondria of plants and fungi, and mitochondria of mammals. Even within these larger groupings, there is a considerable variation in the size and number of ribosomal RNA and protein constituents. At present, our knowledge of the comparative structures of cytoplasmic eukaryotic ribosomes comprises mainly sequence data. The known cytoplasmic eukaryotic small ribosomal subunit RNAs are generally about 1800 nucleotides in length, although extremely large or small ones exist in some organisms. The comparison of many such sequences enables the identification of conserved helical (base-pairing) regions. The proposed secondary structures of small subunit ribosomal RNAs from most organisms are in many ways comparable to that of the E. coil paradigm [19], in that there is a universally conserved structural core which presumably is minimally necessary for proper ribosome functioning. However, there are still many differences in secondary structure of this ribosomal RNA species between prokaryotes and eukaryotes, and also among eukaryotes, that arise within these conserved helices as well as within the less-conserved variable regions. In contrast to the large numbers of ribosomal RNA sequences that have been and continue to be accumulated, there is a paucity of ribosomal protein sequences [20]. No set of cytoplasmic eukaryotic ribosomal proteins has yet been completely defined, although it is likely that there are specific ones unique to eukaryotes. At present, though, interspecies comparisons of ribosome structure are based primarily on ribosomal RNA data. The important question remains how differences in ribosomal RNA secondary structure, as well as diversity in protein composition, are reflected in the three-dimensional structures of these complexes. It has been noted previously that the small ribosomal subunits from many organisms exhibit a greater similarity
in many physical properties than the corresponding large subunits [4,21,22]. It was anticipated at the onset of our studies of Th. lanuginosus ribosomes and subunits that structural differences would be revealed with the aid of electron image analysis. However, alternative approaches such as electron spectroscopic imaging [18,23] or scanning transmission electron microscopy [24] of the ribosomal RNA component selectively, and in situ, may be more efficacious in this regard. Th. lanuginosus excretes amylolytic (starch degrading) enzymes which are of interest for biotechnological applications because of their increased thermostability [25]. The small subunit ribosomal RNA components and large subunit ribosomal RNA genes of this organism have also been examined in the framework of larger investigations on eukaryotic RNA structure and processing [26-29] and RNA-protein interaction [31)1. The presence of conserved A and T rich segments within the generally stabler G and C rich nucleic acids has allowed the identification of domains functionally important in termination and maturation. The ribosomes of this fungus have not otherwise been studied. Because of their strongly conserved eukaryotic structural characteristics and increased thermostability, ribosomal subunits and proteins from Th. lam~ginosus may be attractive candidates for attempts at crystallisation and X-ray diffractometry [31], and thus deserve further investigation. However, standard techniques of electron microscopy alone are of diminished utility in defining characters for phylogenetic categorisation within the eukaryotic kingdom, because of the structural congruity of the small subunit to the resolutions achievable. In summary, we have constructed characteristic projection views of the small ribosomal subunit from the fungus Tit. latmginosus. The averages we obtained corresponded to and appeared ahnost identical to those derived from other eukaryotic species by similar computational approaches. Previous comparative studies of ribosomal subunits from diverse prokaryotic species showed considerable variation in fine structural detail. Our present results provide further evidence that small ribosomal subunit structure is strongly conserved within eukaryotes, despite even the temperature dependence of ribosome function within thermophiles (Ref. 32, cf. also Ref. 33). This knowledge is of significance in consideration of future studies of cukaryotic ribosomal structure and function, showing the latitude to which experimental results can be extrapolated to different species.
Acknowledgements This work was supported by grants from the Research Advisory Board of the University of Guelph. and the Natural Sciences and Engineering Research
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