58

Biochimica et Biophysica Acta, 1132(1992) 58 ~)6 ~ 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.01)

BBAEXP 92401

Electron microscopical projections of the large ribosomal subunit from Thermomyces lanuginosus George Harauz Department ~[ Molecular Biolot,9' and Genetics, Unil,ersityOf Guelph, Guelph (Canada) (Received 28 January. 1992) (Revised manuscript received 8 April 1992)

Key words: Ribosome; Thermophile; Electron microscopy;Image analysis; (Fungus) Multivariate statistical analysis and hierarchical ascendant classification have been used to construct averages of two fundamental electron microscopical views of large ribosomal subunits from the thermophilic fungus Thermomyces lanuginosus. The first was roughly pentagonal in shape and corresponded to the canonical crown view seen in images of large subunits from prokaryotic species. The second and more prevalcnt projection was elliptical in shape, and by matching protuberances could be interpreted as the complex rotated from the crown orientation. Because of its ubiquity and consistency, this elongated view could potentially serve as the standard for structural comparisons of the large ribosomal subunit from eukaryotic organisms, and as the basis for a three-dimensional reconstruction.

Introduction We have previously described the computational analysis of electron micrographs of whole cytoplasmic ribosomes [1] and small ribosomal subunits [2] from Therrnomyces lanuginosus, a mildly thermophilic (55°C) fungus which is of biotechnological interest and whose ribonucleoprotein components are potentially suitable for crystallisation. In the first study, no major morphological differences could be detected between corresponding projection views of fungal and eubacterial (Escherichia coli) monosomes, despite the evolutionary gap between these two organisms [1]. This result could be explained by the relative lack of morphological features of this complex in electron micrographs, as well as potential technical considerations. The later and more computationally exhaustive study of the small ribosomal subunit from Th. lanuginosus yielded averages which appeared almost identical to those derived from other eukaryotic species by similar computational approaches [2], demarcating further the degree to which small ribosomal subunit structure is conserved within eukaryotes (cf. Ref. 3). In this present paper, we describe the results of electron image analysis of the large ribosomal subunit of Th. lanuginosus. To our knowl-

Correspondence to: G. Harauz, Department of Molecular Biology and Genetics, University of Guelph, Guelph, Ontario, Canada, N1G 2WI.

edge, no studies employing computational image analysis of large populations of isolated particles have yet been reported for the eukaryotic complex. (In our own laboratory, we have recently analysed small populations of ribosomal particles from yeast [4].) Here, we define quantitatively two 'standard' views that form a basis for further structural and comparative morphological studies of the eukaryotic large ribosomal subunit.

Electron microscopy of the eukaryotic large ribosomal subunit The overwhelming majority of the literature on ribosome structure is based on studies on isolates from the eubacterium E. coli. Nonetheless, over the past few decades there have been many investigations on ribosomes from eukaryotic organisms. In order to set the context for the work described in this current paper, it is important first to review some of the literature that pertains primarily to electron microscopy of the large subunit from eukaryotes. There are other reviews that encompass discussion of electron microscopical investigations of the eukaryotic small subunit as well as of the whole monosome [5-8]. One of the earliest structural studies was that of Haga et al. [9] on rat liver ribosomes. In micrographs of preparations negatively stained with uranyl oxalate, the large subunits appeared as round or slightly elongated structures, of dimensions 24.4 _+ 1.7 nm × 20.7 _+ 1.8

59 nm. The intact large subunit appeared to contain a solvent-filled groove, or channel, but which was not as clearly visible as in E. coli. A year later, Nonomura et al. [10] described the electron microscopy of negatively stained (with uranyl acetate) preparations of monomeric ribosomes and both subunits from rat liver. In particular, the large subunit exhibited a systematic predominance of a few types of images: (i) rounded (of about 23 nm in diameter); (ii) a 23 nm long asymmetric 'skiff', with two convex sides, and with one flattened or concave side, showing a notch about 4 nm towards the blunted end of the particle; and (iii) a more equilateral triangular type. The phenomena of the particles exhibiting only a few characteristic profiles, and of relatively few enantiomers of the asymmetric ones, were surmised to be due to rapid binding to the carbon support via hydrophobic interactions. Examination of images of tilted specimens showed that there was a finite range of particle positions within the same general type of image. The rounded profiles were interpreted to have a flat face which abutted the small subunit, and which had a depression filled by stain. Kiselev et al. [11] studied rat liver subunits by 'visual processing of the micrographs', during which the authors 'selected images of particles that they believed to be most typical due to their frequent recurrence', complemented by analysis of tilted images and the successive refinement of plastic models. The main views discerned were (i) a rectangular one, 15 nm x 22 nm x 24 nm in size, and (ii) a lateral one, 'rods' of size 22-34 nm x 7-9.5 nm, with a 'channel' about 8 nm x 5-6 nm. The large subunit was described as a flattened body with a channel on one side and two protuberances (one large and one small) that could elicit the preferred orientations. The channel was speculated to represent a kind of 'RNA center', and this and the protuberances were situated on the same side of the large subunit that faced the small subunit within the complete monosome. Boublik and Hellmann [12] compared the structures of ribosomes and subunits from E. coli ('the classical prokaryote') and the brine shrimp Artemia salina ('a convenient eukaryotic source') by electron microscopy of negatively stained preparations. The eukaryotic large subunits were rounded particles, of diameter 26 n m + 10%, with no obvious preferential orientation. The E. coli large subunits were 22.5 n m + 10% in diameter, but with a highly preferred crown orientation. Thus, the structural comparison of this complex from the two species had to be restricted to the small subset of crown views that could be selected from the eukaryotic micrographs. The crown view of the prokaryotic large subunit was characterised by the three crests now known to be the E L 7 / E L 1 2 stalk, the central protuberance, and the ELI protuberance, or shoulder. (Here, the 'E' refers to E. coli ribosomal proteins.) The eu-

karyotic large subunit also had a middle crest (central protuberance) and a left crest (stalk) with a variable orientation. The right crest (Ll-analogous protuberance) appeared rounder in the eukaryote, which was suggested to explain, in part the lack of an obvious preferred orientatioti of the complex. The eukaryotic large subunit also showed a knob-like protrusion, about 4 nm X 5 nm in size, at its base, which was suggested to be the site of attachment to plasma membranes. In a subsequent study [13] on ribosomes from the slime mould Dictyostelium discoideum, the large subunit was found to be less than 25 nm in size, and to be less complex than that of A. salina, appearing as an oval particle with only a single protrusion. Boublik et al. [3] have found, on the whole, that whereas the appearance in electron micrographs of small subunits from phylogenetically distinct eukaryotes appeared highly conserved, the large subunits showed greater structural medley. In a series of systematic visual comparisons of the appearance of ribosomal structures from diverse species [14-18], it was inferred that the structures of large ribosomal subunits from eubacteria and most archaebacteria were very similar, but that those from sulphur-metabolising archaebacteria ('eocytes') and eukaryotes presented three additional features: a bulge at the region of the Ll-analogous shoulder, a lobe at the right side of the base, and a gap (albeit filled in eukaryotes) between the two. The stalk appeared to be a universally conserved feature in ribosomes from all organisms. As part of their investigations on localising modified bases within ribosomal RNA, Montesano and Glitz [19] described the structure of wheat germ cytoplasmic ribosomes and subunits by electron microscopy of negatively stained preparations. In particular, the micrographs obtained of the large subunit were of excellent quality and represented a significant achievement. Visual inspection of large numbers of micrographs allowed the extraction of a number of similar views of the large subunit, most of which were variations of the 'quasi-symmetric' (crown) type with three protuberances, but an 'asymmetric' (kidney) form was also noted. The three protuberances corresponded to the stalk, central protuberance, and the shoulder. When present, the stalk exhibited different orientations (within a 45 ° range) with respect to the main body of the subunit. When the stalk was absent (probably lost during the preparation procedure), the particles appeared more or less symmetrical about a vertical axis. Another major dissimilarity amongst different subunits was in the depth of penetration of stain between the central protuberance and shoulder. Finally, quasi-symmetric forms that were squatter, i.e, more elongated horizontally, were also seen. The overall conclusion presented on this part of the study was that the wheat

6(I germ large ribosomal subunit resembled the paradigmical E. co# one in overall shape, although with a deeper notch and sharper angle between the central protuberance and body. Most recently, Verschoor and Frank [20] reconstructed in three dimensions the cytoplasmic ribosome from rabbit reticulocytes. Of note here is the description of the appearance of the 60S subunit within the monosome reconstruction. (The isolated 40S subunit had previously been reconstructed [21].) The 60S portion was in close contact with the 40S portion at the base. The former had an ellipsoidal shape, with the major axis oriented at about 45 ° from the vertical defined b y the line joining the central protuberance and the base. This elongated shape was more subdued when the reconstruction was viewed from certain directions, and then the more rounded appearance was similar to that of the eubacterial (E. coli) structure. At one end of the complex was a central protuberance, a small shoulder akin to the ELI protuberance in the E. coli 50S subunit, and a stalk, albeit not as extended as the E. coli E L 7 / E L 1 2 one. A protruding ridge ran horizontally along the exterior face, i.e., the outside surface of the 60S portion within the monosome. On the interface side was a 'canyon' analogous to that observed in the E. coil 50S subunit. Verschoor and

Frank [20] concluded that the eukaryotic large ribosomal subunit matched the eubacterial one fairly well, within limitations of the methodology, and that its extra mass could conceivably result in a different shape. In summary, then, the work that has hitherto been reported on electron microscopy of eukaryotic large ribosomal subunits has revealed a gross morphology akin to that of the eubacterial complex. Due to the difficulty of purifying these ribosomal structures without degradation, and to the inherent limitations of biological electron microscopy, finer structural features varying within and possibly unique to this kingdom have not yet been clearly defined. One approach that has significantly advanced our ability to probe ribosomal structures has been computerised image analysis of electron micrographs of purified particles, to construct averages with an increased signal to noise ratio of their characteristic (recurring) projections [22-24]. We have previously described the characteristic views of whole ribosomes and the small ribosomal subunit from Th. lanuginosus as determined by such an approach [1,2]. This report continues the prerequisite first stages of these studies on the Th, lanuginosus ribosome, viz., the quantitative assembly of images of preferred orientations of the large subunit as seen in negatively stained preparations. Materials and Methods

Fig. I. Electron micrograph of a negatively stained preparation of large ribosomal subunits from Th. lanuginosus. Recognisable crown (c) and elongated (e) projections are circled, but some are unlabelled for the reader to classify. The arrows point to a peculiar double horned view. Scale bar represents 100 rim.

The growth of Th. lanuginosus and isolation of ribosomes and ribosomal subunits were performed as previously described [2]. Purified large ribosomal subunits were prepared for electron microscopy by adsorption to a carbon film and negatively contrasted with 2% uranyl acetate. Electron micrographs were taken on a J E O L JEM-100CX at an instrument magnification of 50000 x and an accelerating voltage of 80 kV. Each specimen area was not preilluminated prior to being micrographed, to minimise the total electron dose. Six electron micrographs were digitised using an Optronics rotating drum densitometer and a scanning step size of 25 p.m, corresponding to 0.5 nm at the object level. Digitised images were transferred via magnetic tape to an old IRIS 3120 workstation (Silicon Graphics, Mountain View, CA). A total of 830 distinct particles were selected interactively, i.e., subimages of size 100 x 100 pixels and containing a single ribosomal subunit within them were extracted from the larger micrographs. The image set underwent single particle data analysis in order to construct averages of the predominant characteristic views [1,2,24]. The initial step was a bandpass filtering operation to emphasise intermediate spatial frequency detail, followed by normalisation of the grey levels. Then the mechanisms of multi-reference alignment (MRA), multivariate statistical analysis (MSA), and hierarchical ascendant classifi-

61 cation (HAC) were applied iteratively to the entire population of images and then to more homogeneous subpopulations as previously described [2]. Results A field of view of negatively stained large ribosomal subunits from Th. lanuginosus is shown in Fig. 1. Both rounded and elongated complexes are observed, and are of the order of 25 nm in size. The computational analysis of electron micrographs of 830 ribosomal particles from Th. lanuginosus enabled the grouping together of those subunits lying in the same orientation on the support film, and the rejection of other images that were atypical. Images within each group could then be averaged together to give a picture in which noise, due to factors such as variable stain distribution (especially along the periphery of each particle) and an uneven carbon background, was reduced with respect to the common structural signal (Fig. 2). At each cycle of the analysis, the use of new averages as references led to a better overall alignment of the data set, and to a better definition of homogeneous subgroups. The first eigenimage derived by multivariate statistical analysis and describing the principal component of varia-

¢

tion of the data set accounted for progressively more and more of the interimage variation as the cycles proceeded: 15.6%, 19.3% and 35.6%. These figures indicate an increasing homogeneity of the data undergoing correspondence analysis [25], an effect achieved mainly by the better by the better alignment of individuals within the population, thus facilitating their comparison. The population of 830 images of the large ribosomal subunit exhibited two recognisable and recurring motifs. The first one was roughly pentagonal in shape, and corresponded to the canonical crown view typically seen in preparations of this complex from prokaryotes [26]. Averages of this view are shown in Fig. 2a-g, and an average with contours superimposed is shown in Fig. 3a. The second and predominant preferred mode of orientation was an elongated and asymmetric one with a major protuberance pointing towards the top, and often a plume-like protrusion at the left (Fig. 2h-o). Within the starting set of 830 images, 449 were well-aligned and found to lie in this orientation - these were selected out for a separate cycle of the single particle analysis. Fig. 3 b - f show specific averages constructed after this stage to show selected morphological details, including the plume-like protrusion at the left,

d

e

~iiill

h

O

o

Fig. 2. Averages of images of classes of Th. lanuginosus large ribosomal subunits chosen on the basis of internal homogeneity, i.e., a relatively low intraclass variance. The class averages constructed here have a reproducible spatial resolution of the order of 2.5 nm. (a-g) Crown projection. (h-o) Elongated, asymmetric projection. The number of particles within each class is (a) 33, (b) 29, (c) 20, (d) 33, (e) 32 (f) 26, (g) 22, (h) 44, (i) 53, (j) 29, (k) 20, (1) 32, (m) 30, (n) 54 and (o) 34.

62 and a groove running down the middle of the complex towards the right.

Discussion Sucrose density gradient centrifugation was used to p u r i f y l a r g e r i b o s o m a l s u b u n i t s f r o m Th. lanuginosus, It

has previously been noted that eukaryotic ribosomal s u b u n i t s a r e e s p e c i a l l y l a b i l e t o h a n d l i n g [9,13,19]. Here, the amount of handling of the particles was m i n i m i s e d , a n d i s o l a t i o n c o n d i t i o n s w e r e s u c h as to i n h i b i t d e g r a d a t i o n by e n d o g e n o u s e n z y m e s . T h e b e s t c h a r a c t e r i s e d v i e w in e l e c t r o n m i c r o g r a p h s of the large ribosomal subunit has been termed the

Fig. 3. Averages of subpopulations representing reproducible modes of adsorption to the carbon support of the large ribosomal subunit of Th.

lanuginosus. (a) Crown view (average of 16 images), for comparison with corresponding view of prokaryotic complexes. Peripheral structural features are marked as follows: +s' = L7/L12-analogous stalk, 'c' = central protuberance, 'n' = notch, 'l' = Ll-analogous shoulder (or right crest), g = +gap', 'b' = basal lobe, +i' = basal incision. The two solid arrows point to two potential lateral incisions on the left-hand side. (b) Average of a large number (165) of particles lying in the elongated, asymmetric orientation. The labels 's'+ 'c' and "1' are as in (a). (c) Construction o1( elongated, asymmetric view with the central groove pointed to the top (average of 4 images). (d) Construction of elongated, asymmetric view with lhe central groove pointed to the top, and with a prominent plume-like protrusion at the left (average of 5 images). (e) Construction of elongated, asymmetric view with the central groove in the middle (average of 19 images). (f) Construction of elongated, asymmetric view with the central groove tending towards the bottom (average of 20 images). The scale bar represents 10 nm.

63

Stalk (EL7 / EL12 dimer)

Central protuberance j

Diagonal groove Ridge or Shoulder

Split

Lateral incision

-

-

Gap (?) Basal lobe (?)

/ Basal incision

Fig. 4. Schematic illustration of features seen in enhanced electron micrographs of large ribosomal subunits from the prokaryote E. coil (redrawn from Ref. 26). In this paradigmical large subunit, the stalk comprising dimers of the ribosomal proteins EL7 and ELI2 was strongly stain excluding. Below the stalk on the left hand edge was a bright spot of stain exclusion, and below that a shallow lateral incision. The central protuberance was rounded and quite bright, and directly below it was a heavily stained groove extending diagonally across the subunit. At the base of the particle was a stain incision pointing to the right. The most heterogeneity of appearance of E. coil crown averages was on the right hand side in the region of the protuberance containing ribosomal protein EL1; this protuberance either formed a protruding shoulder or was split. Crown views from the archaebacterial species also predominated and bad the same prongs of the crown, formed by the L7/L17-analogous stalk, the central protuberance, and the Ll-analogous protuberance. The most variability in crown view appearance amongst various prokaryotes was on the right hand side of the particle, although the stalk and central protuberance also showed differences in brightness and shape. crown view, whose characteristic structural features are shown in a schematic in Fig. 4. From the partition of the entire data set of 830 particles, the set of best crown class averages (in terms of number of members and degree of internal homogeneity) is shown in Fig. 2a-g, comprising 195 particles. The particles are oriented in these images with the prongs of the crown pointing towards the top, but there is a considerable amount of variation in finer structural features. In Fig. 3a, contour lines were superimposed on an average constructed from a selected homogeneous subset, and specific attributes are demarcated more explicitly for reference in the following discussion. The LT/L12analogous stalk appears to be present at the left, but indistinctly. On either side of the base of the stalk are two strongly stain excluding foci. Travelling clockwise along the periphery, the central protuberance is also stain excluding. The entire region comprising these features forms a double-horn type of arrangement. Thus, the unusual bilobed structures observed in Fig. 1 are probably variations of the crown projection. Travelling further clockwise from the central protuberance, there is a strong indentation of stain, forming a notch. The Ll-analogous shoulder appears different in the different class averages. It sometimes juts out with a

hint of a wispy appendage, but in other instances appears sharply attenuated. Below this feature on the right-hand side are a gap, a basal lobe, and a basal incision. There appears to be another basal lobe to the left of the incision, and one or two gaps below the stalk. Within the interior of the particle is a heavily stained groove running from above the Ll-analogous shoulder to below the base of the stalk, as well as two or three other foci of stain. Such a degree of variation of structural details between different classes is not unexpected, and can be explained by the vicissitude of peripheral stain deposition and penetration (including possibly positive staining of exposed ribosomal RNA), as well as by the rocking of the particle. These phenomena are not yet well-understood, given our limited knowledge of the molecular composition of these complexes and of the physico-chemical processes involved in their interaction with uranyl salts. Previously, electron image analyses of large ribosomal subunits from various prokaryotes yielded averages of the crown orientation which could be used for interspecies comparison [24,26]. The construction of one of the crown views of Th. lanuginosus (Fig. 3a) can thus be compared directly with those of prokaryotes. In the fungus, the stalk on the left hand side is present but not especially large. This result was due to the flexibility of this structure in individual images, making it indistinct in the average. The central protuberance is distinct but with a bilobed appearance. The right crest (Ll-analogous protuberance) forms a distinct ridge with a significant amount of stain penetration between it and the central protuberance. There is a hint of a split on this protrusion. Travelling further clockwise along the periphery of the particle, there is a shallow gap, and then two basal lobes with a notch between them. On the left hand side are two lobes of stain exclusion. Within the particle, a stain-filled groove extends diagonally from the central protuberance to the left side below the stalk. The Th. lanuginosus crown projection, then, differs from that of E. coli in appearance of most of the peripheral features, and in that the diagonal groove lies slight higher up in along the particle. These differences in appearance are of the same degree of severity as those seen between crown views from archaebacteria, and their definition is thus reasonable. The appearance of the Th. lanuginosus crown view is consistent with that of yeast, in which the basal lobe and L7/L12-analogous stalk were identified [16], and in wheat germ [19]. This good agreement with the present results supports the development of a consensus model for the eukaryotic large ribosomal subunit [4]. The most prevalent orientation in electron micrographs of Th. lanuginosus large ribosomal subunits is an elongated one, averages of which are shown in Fig. 2h-o, comprising 296 particles. There are two major

64 protuberances at one end of the complex, a strongly stain excluding one pointing upward, and a less prominent one extending towards the left. In Fig. 21, this protrusion presents a plumelike aspect. Besides the two major protrusions at the left end of the particle, each class average has two stain-excluding (white) streaks joined at the center, and running roughly parallel to one another from left to right, and defining a large groove. Towards the right of the top protuberance is a stain incision that varies in depth, occasionally appearing to extend into a small groove that traverses the particle towards the left-hand side. Travelling further clockwise along the periphery are one small and one intense foci of stain exclusion. In some classes, there is the hind of an appendage extruding upwards (Fig. 2m-o). The horizontal groove extends to the periphery of the particle. As one scans Fig. 2 h - o in sequence, the particle appears to get somewhat fatter. In Fig. 2h-j, the bottom edge of the structure is distinct, becoming less sharply demarcated in Fig. 2k-o. In order to understand this elongated, asymmetric view better, a subpopulation of 449 large ribosomal subunits presenting this aspect was analysed alone in a fourth cycle of M R A / M S A / H A C . A subset of 165 images showing this orientation was then extracted from the larger data set for more detailed examination, and class and specific averages are shown in Fig. 3b-f. Exceeding care had previously been taken to omit from the analyses any small subunits in the right-lateral orientation that were found infrequently in the preparation. Rocking of the particle around this preferred mode of adsorption was suggested since the groove could be seen closer to either the top or to the bottom, when it gradually became obscured by the light areas bounding it (Fig. 3c,d). The lability of the particle in this orientation is suggested by the two class averages in Fig. 3e and f, in which the horizontal groove appears to shift upward. In light of previous studies on the eukaryotic large subunit [19], and in comparison with the crown view averages constructed here (Figs. 2a-g, 3a), we interpret the leftward pointing plume to represent the L7/L12analogous stalk, and the upward pointing protrusion as being the central protuberance (Fig. 3b). The region towards the right of the putative central protuberance comprises a notch extending into a diagonal groove, and foci of stain exclusion that appear to correspond to the Ll-analogous shoulder (Fig. 3b), even with the occasional wispy appendage (Fig. 2a-c, m-o). The major horizontal groove evident in this projection extends to what may be the basal notch. Other peripheral details may be matched together between the crown and elongated projections, although it may be premature to do so. It was pointed out by a reviewer that the plume in Fig. 3d has an appearance very similar to the E L 7 / E L 1 2 stalk of E. coli [27]. In both structures,

there are two stain excluding maxima, the distal one being a fan-shaped blur and presumably joined to the proximal one by a flexible hinge. Variations between different class averages of the elongated as well as of the crown view are due to the variability of definition of the particle boundary by the negative stain, an effect exacerbated by rocking of the particle. This latter phenomenon was noted by Nonomura et al. [10], and has been studied quantitatively with other ribosomal subunits (cf., Ref. 23). On the bases of this knowledge and the current results, it is suggested this elongated asymmetric view is not a single projection as such, but rather a mode of adsorption about which the particle may rock, exhibiting a finite range of orientations. The appearance of a macromolecular complex in electron micrographs of negatively stained preparations depends on a myriad of factors relating to overall morphology and chemical composition, which influence the modes of adsorption of the particles to the carbon film, and their interaction with negative stain. Nonetheless, the averaged images presented here agree well with the early visual analyses of micrographs of the large ribosomal subunit from eukaryotes, and illuminate some aspects. One of the interesting considerations of the work by Kiselev et al. [11] was their construction of a boxy model of the eukaryotic large subunit. The results presented here in Figs. 2 and 3 suggest that the interpretation by Kiselev et al. of rectangular projections was caused by the numerous appendages, which may appear to form sharp corners in views of individual particles. Furthermore, the micrographs of eukaryotic large subunits presented by Boublik et al. [3,8,12,13], and Montesano and Glitz [19] were presented with an upward pointing protrusion that appeared to vary in location from the left to the right of the particle. The current results suggest that this protrusion is usually the central protuberance, and that its apparent movement from the center to the left is due to its changed appearance in distinct projections, one crown and the other elongated. Nonomura et al. [10] and Montesano and Glitz [19] elucidated two forms (presumed to be enantiomeric) of an elongated, asymmetric view, called the 'skiff' in the former paper. In recent work on the computational analysis of small populations of large ribosomal subunits from yeast, a 'long-crown' view was extracted and averaged [4]. The basic shape of the subunit in this orientation was that of a thick 'L', with one prominent protrusion (the base of the 'L') and a groove spanning 7.5 nm between it and the main body. These features could be interpreted as representing the El-analogous protuberance and the diagonal groove as visualised in the crown orientation. We suggest that the yeast longcrown and the Th. lanuginosus elongated view are not enantiomers, since the protuberances in each (which

65 point in opposite directions) could represent different protein moieties. Here, in the elongated and asymmetric view, the main protrusions are interpreted to be the LT/L12-analogous stalk and the central protuberance. We propose that this view be the 'skiff', with the stalk being the rudder and the central protuberance being an elevated stern. The predominant negatively stained grooves in both the crown and skiff views (Fig. 3a,b) are probably different according to our ideas of the orientations that are represented. However, now there is a morphological framework for localising this feature, as well as for defining more precisely the number and nature of protuberances visible in different views. The overall shape of the eukaryotic complex in the crown projection can be related directly to that of prokaryotes. This observation is compatible with the concept of preservation of ecumenical structure of a complex involved in a particular function. The small subunit in both prokaryotes and eukaryotes is involved in messenger RNA and transfer RNA recognition and binding, and the large subunit catalyses primarily the reactions of polypeptide elongation. Nonetheless, it is known that the eukaryotic large subunit is bigger and more intricate than that of prokaryotes, in both RNA and protein composition [28-30], and the current results are consistent with there being many subtler structural differences between eukaryotic and prokaryotic ribosomal subunits. Even though our present knowledge of details of the constitution, biogenesis and involvement in translation of eukaryotic ribosomes is relatively sparse, it is worthwhile to consider biological reasons why eukaryotic and prokaryotic ribosomal subunits are structurally distinct. A greater structural complexity can in part be explained on a molecular evolutionary level: eukaryotes have lacked the selective pressures that have kept prokaryotic genomes more streamlined. Consequently, larger ribosomal RNAs and more ribosomal proteins have resulted by gene duplication and elongation events, and mutations in posttranscriptional processing. As long as the overall function of the ribosome as an enzymatic complex involved in protein synthesis was not adversely affected, such changes could be retained. The basic mechanics of protein synthesis are common to both kingdoms, which is why the basic shape of ribosomal subunits is the same. On the other hand, ribosome biogenesis is more complicated in eukaryotes than in prokaryotes, involving both posttranscriptional and posttranslational modifications of the constituents, the transport of ribosomal proteins through the nuclear envelope for assembly in the nucleolus, and the return to the cytoplasm of complete subunits. Messenger RNA in eukaryotes has a significant amount of inherent secondary structure and is packaged as a ribonucleoprotein complex [31]. Translation in eukaryotes thus requires concomitant

processes of disrupting secondary structure (particularly double-stranded helical regions) and exogenous protein binding. It has been recently shown in yeast that within the spb class of genes, one codes for a ribosomal RNA helicase while another codes for ribosomal protein YL46 [32]. Moreover, the protein that binds to the poly(A) tail of eukaryotic messenger RNA also binds to the large ribosomal subunit [33]. Thus, there is an intimate involvement of the eukaryotic large ribosomal subunit with constituents of the messenger ribonucleoprotein particle, and some of the subunit's proteins might possess helicase activity. It is also interesting to speculate whether or not eukaryotic ribosomes play a role in translational control of gene expression. Finally, the attachment of ribosomes to internal membrane systems such as the nuclear envelope and the endoplasmic reticulum for purposes of cotranslational passage may be mediated in part by specific receptors [34]. Boublik et al. [12] identified a large protrusion in the base of A. salina subunits imaged in the crown projection, which they postulated could represent a membrane attachment site. In the present work on Th. lanuginosus ribosomes, a basal lobe is observed to the left of the basal incision (Fig. 3a), which could correspond to the A. salina protrusion, even though the latter is much larger. In summary, eukaryotic ribosomes have a potentially greater functional diversity than prokaryotes, which should be reflected in their structure. Here, we have constructed average views of two characteristic electron microscopical projections of the Th. lanuginosus large ribosomal subunit, which form a basis for ensuing structural and morphological studies of this eukaryotic complex.

Acknowledgements This work was supported by an operating grant from the Natural Sciences and Engineering Research Council of Canada. I am grateful to Mr. Derrick Flannigan for ribosome preparation, Dr. Patrick Whippey (University of Western Ontario) for support with the densitometry, and to Mr. Harry Zuzan for assistance with the image processing.

References 1 Harauz, G. and Flannigan, D. (1990) Biochim. Biophys. Acta 1038, 260-267. 2 Harauz, G. and Flannigan, D. (1992) Biochim. Biophys. Acta 1130, 289-296. 3 Boublik, M., Hellmann, W. and Jenkins, F. (1982) Proc. 10th Intern. Cong. Electron Microsc. (Hamburg) 3, 95-96. 4 Kyle,K.M. (1991)Electron microscopicalinvestigationsof ribosomal particles from Saccharomyces cererisiae, M.Sc. Thesis, University of Guelph, Canada. 5 Lake, J.A, (1983) Prog. Nucl. Acad. Res. 30, 163-194. 6 Lake, J.A, (1985) Annu. Rev. Biochem. 54, 507-530. 7 Frank, J., Verschoor, A., Radermacher, M. and Wagenknecht, T.

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