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TIBS - October 1983 of magnitude faster conduction than the Grotthus type of proton transfer mechanism that is operative in solutions. Even il an extensive intermembrane space were to exist to accommodate the bulk aqueous phase, the aforementioned mechanism of proton conduction could still be operative. While a considerable body of evidence" exists on the 'structured' water in biological systems, the functional relevance of such a water remains to be explored. Clearly there is a need to ascertain the real structure of

References

5 Kell, D. B. (1979) Biochim. Biophys. Acta 549, 55-99 6 Finney, J. L. (1979) Water - A Comprehensive Treatise, Vol. 6 (Franks, F., ed.), pp. 47-122, Plenum Press S. K. MALHOTRA

1 Williams, R. J. P. (1983)Trends Biochem. Sci. 8, 48 2 Van Harreveld, A., Crowell, J. and Malhotra, S. K. (1965)J. Cell Biol. 25, 117-137 3 Malhotra, S. K. and Van Harreveld, A. (1965) J. Ultrastruct. Res. 12. 473~1.87 4 Malhotra, S. K. (1966)J. Ultrastrua:t. Res. 15, 14-37

and s. S. SIKERWAR Biological Sciences Electron Microscopy Laboratory, Department of Zoology, University of Alberta, Edmonton T6G 2E9, Canada.

mitochondria, and other energy transducing organelles, in relation to the mechanism (s) of energy transduction.

Reviews A consensus model of the Escherichia coli ribosome Jeffrey B. Prince, Robin R. Gutell and Roger A. Garrett In this article the latest results are summarized on the localization o f proteins, RNA sites, and various ligands on the ribosomal subunits o f E. coli. For the proteins, the data derive primarily from two kinds o f experimental approach." neutron scattering o f reconstituted subunits, containing pairs of deuterated proteins, which yields both the distances between centres o f mass o f the two proteins and their radii of gyration, and immune electron microscopy which visualizes protein-bound immunoglobins (IgG) on the ribosomal surface. Although the two approaches yield different kinds o f data, the results are integrated into a consensus model partly because the level of agreement between the methods is good. For the RNA and ribosome-bound ligands the data are available exclusively from the immune electron microscopy method. Success in applying the immune electron microscopy (IEM) technique to ribosomes, from which most of the data originate, depends on the proteins (and RNA) having accessible antigenic determinants on the ribosomal surface. This was first demonstrated for the 30S subunit proteins I and later for the 50S subunit proteins by using a variety of immunochemical and physical methods. Most of the protein-specific IgGs produced subunit-IgG-subunit complexes ('dimers') which facilitated the protein localizations. The latest protein results from the neutron scattering and IEM methods derive from three main groups: Moore et al. 2"3 (Yale), Lake et al. 4"~ (UCLA) and St6ffieret al. 6 (Berlin) for the 30S proteins, and the latter two groups for the 50S proteins. The RNA data come, in addition, from the laboratories of Vasiliev (Poustchino) and Glitz (UCLA). Our crite-

Jeffrey B. Prince and Robin R. Gutell are at the Thimann Laboratories, University o f California, Santa Cruz, CA 95064, USA. Roger A. Garrettis at the Department o f Biostructural Chemistry, Kemisk lnstitut, Aarhus University, Denmark.

rion for including a site in the model is that two, or more, laboratories agree on a particular location, although this criterion is not always rigorously followed for the 50S subunit proteins where the data are scarce. The use of unpublished results for the assignments has generally been avoided, although some revised protein locations from the Berlin group are available only in meeting abstract form with minimal experimental data. Results from other approaches, including chemical crosslinking, 'affinity labelling' of functional sites, and fluorescence energy transfer, are only invoked when the evidence is particularly good (e.g. a high yield of chemical cross-linking), or when there is general agreement amongst the biochemical results.

and a thin projection or 'platform', tilted towards the subunit interface4. The Poustchino model, obtained by shadowing, is similar except that no cleft is observed between the body and the platform ('ledge'), and the body is segmented into upper and lower halves7~. The Berlin model originally contained a large body and a smaller head region with two symmetrical lateral lobes which pointed into the subunit interface thereby producing a hollow in the centre of the ribosome. One of the lateral lobes has now been enlarged, thus producing an asymmetric model, and the 'hand' has also been changed such that it resembles the other two models more closely. Recently, Korn et al. "~have derived a model from dark field electron microscopy with improved resolution ( - 15 /~), compared with the aforementioned studies ( - 2 0 ,~); it approximates to that of the Poustchino group. They also suggest that the apparent cleft in the UCLA and Berlin models may be due to positive staining of RNA in that region. We have chosen the UCLA model in this study only because it has proved the least variable of the two main IEM models, but we express no opinion as to its relative accuracy. The UCLA model of the 50S subunit consists of a large lower body with a large central protuberance lying between a smaller protuberance and a stalk-like structure projecting from the body (see Fig. 4). The early Berlin model was similar except that it was a pseudosymmetrical structure lacking the 'stalk'. Recently, a few laboratories employing different electron microscopy methods have established that the large subunit is indeed asymmetrical and that the ' stalk' does exist'" to.

Shapes of the ribosomal subunits Although differences persist in the published models, there is now general agree- 30S subunit Protein sites. The 30S subunit protein ment on the overall structure of the 30S subunit. The UCLA model, obtained by locations, depicted in Fig. 1, are classified negative staining, consists of a large'body' into three groups. Sites drawn with closed (lower 2/3), a smaller 'head' (upper 1/3) circles are the most reliable. The broken

~) 1983. ElsevierScience Publishers B.V., Amsterdam 0376 - 5067/83/$01.0()

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b.

1,18

EXTERI 0 R

I N TER FACE

Fig. 1. Consensus model of 30S subunit. (a) exterior surface; (b) subunit interface. Circles designate positions of protein and RNA sites on two-dimensional projections of each subunit surface. Closed or broken circles are site locations with greater or lesser certainty, respectively. Brackets indicate region of site location of proteins in margin (surface unspecified), mRNA - entry/exit site of mRNA; P m = puromycin. Subunit

shape afterLakeand co-workers'. circles indicate less certain sites; for example, they may lie intermediate between two close sites assigned by different groups. Finally, proteins that have been localized within a specific section of the subunit, but at different positions, are listed adjacent to that section of the model. While the protein sites in Fig. 1 depict surface locations (with the possible exceptions of $4 and $8), the neutron scattering data yield the relative positions of the centres of mass of the proteins. To emphasize this distinction, the 12-protein model derived by Moore and co-workers 2.3 is also shown (Fig. 2); the view is apparently equivalent to that of the exterior surface presented in Fig. 1a. Locations are specified for thirteen proteins in our map: $3, $4, $5, $6, $7, $8, $9, SI0, SI1, S12, S13, S14 and S15. In addition, proteins SI, S18, and S19 are assigned to general regions. Although $6, S l l , and S 13 are exposed on both the exterior surface and the interface side of the subunit, these locations probably correspond to one site on the subunit. Protein S19 is the only protein currently assigned to two widely separated sites ( - 1 0 0 /~ apart) by the UCLA group, and although the Berlin group agrees that it lies in the upper part of the head", their positioning does not coincide with either of the UCLA sites. In general, the shapes of the proteins, within the ribosomal subunits, approximate to globular structures; of twelve proteins that have been examined by neutron scattering only two (S 1 and $4) have yielded gyration radii that are incompatible with globular structures 2'3. Our confidence in the consensus model is reinforced by other structural and func-

ticular disagreement may, therefore, reflect differences in either the antigenic specificities of their immunoglobulins or the structural state of their isolated ribosomal subunits. 1 6 S R N A sites. Parts of the 16S RNA structure have been mapped on the 30S subunit by using antibodies raised against either haptens covalently attached to one of the termini of the RNA chain or naturally occurring modified nucleotides. Using the former approach, three groups have localized the 3'-end of the 16S RNA at approximately the same position on the upper platform 7'11,12 (see Fig. lb); the Berlin group also demonstrated that subunits, reconstituted with the derivatized RNA, are active in the formation of an initiation complex with R17 mRNA TM. A dinitrophenyl hapten, attached to the 5'-terminus of the RNA, has also been located by the Poustchino group in the lower body a although, in the absence of any supporting evidence, this result is considered tentative. The large distance between the 3'- and 5'-ends of the 16S RNA (> 100/~) suggests that a major conformational rearrangement occurs after processing the 17S RNA precursor when the two ends are presumably adjacent. Two N",N'Ldimethyladenosine residues (rn~ A) which occur about 25 nucleotides from the 3'-terminus of 16S RNA have been mapped with antibodies raised against the modified nucleoside 13. The specificity of the antibody reaction was established by showing that no IgG would bind to subunits isolated from a kasugamycin-resistant strain ofE. coli that lacks m~ A. Consistent with the location of the 3'-end, the m~ A residues have been placed on the lower platform (Fig. 1b). Another minor nucleoside, 7-methylguanosine (m7G), which occurs at position 526 in E. coil 16S RNA, lies at the junction of the upper body and head (Ref. 14 and Gutell, R. R., Politz, S. M., Meredith, R. D., Erlanger, B. F. and Noller, H. F., unpublished results). (The

tional evidence, and in particular the chemical cross-linking data. In addition to S13 and S19, three pairs of adjacent proteins, $5--$8, $6-S18 and $7-$9 have been obtained in high yields in several laboratories using different chemical reagents; the model is clearly consistent with these results. Of these six proteins all but S18 and S19 are located. The Berlin group locates S 18 close to $6 '~and the Yale group reports preliminary evidence for an undefined position neighbouring $62; we have placed it, in brackets, in the upper body region of the subunit. In addition, although the UCLA and Berlin groups disagree on the precise location of S19, they nonetheless both place this protein close to S13 ~'". Further structural evidence in support of the protein sites derives from chemical and photoaffinity labelling studies; proteins labelled by analogs of tRNA and mRNA which were pre-bound to the ribosome tend to cluster in the head and upper body, respectively (discussed below). The functional and assembly evidence is less compelling because proteins related by function or during assembly need not be physically close. Nevertheless, the three proteins $4, $5 and S12 that can incur mutations which alter the ribosome's response to streptomycin and, therefore, the accuracy of translation, are all clustered in the upper body. Recently, the UCLA and Berlin groups have reported that proteins $45 and $86, respectively, are not available for antibody binding on the ribosomal surface. How- Fig. 2. Neutron scattering model of 30S subunit ever, the former group has localized $84 proteins. Proteins are shown as spheres of the and the latter, $4 (on both E. coli and appropriate volumes; S12 is behind $5. AfterMoore B. stearothermophilus subunits) 6. This par- and co-workers2.

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same result was also obtained for the mTG at position 474 in a chloroplast 16S RNA 14.) In addition to the sites directly visualized by antibody labelling, the position of some RNA regions can be inferred from the locations of the proteins which associate with them. One of the secondary structural models (Fig. 3) which has been proposed for E. coli 16S RNA by Noller and Woese ~5 is based upon chemical, enzymatic and phylogenetic evidence. Numbers indicate the approximate RNA binding site of the corresponding protein as determined by ribonuclease protection or photochemical cross-linking studies. From the known protein-RNA associations, it is possible to place domain II of the RNA on the left side of the upper body (in the exterior view of the UCLA model) and domain III in the head of the subunit. In addition, the lower surface of the 30S subunit, and the subunit interface region, appear to be particularly rich in RNA 2 (see also Fig. 1). Of course, our understanding of the RNA organization is still very limited and will only improve as more tertiary interactions are defined. Ligand binding sites. The binding of a few ligands, including tRNA and mRNA, have been ascertained on the 30S subunit either directly or indirectly. AfFinity labelled puromycin, an inhibitor of peptidyltransferase, can be cross-linked to both ribosomal subunits by ultraviolet light, with Sl4 the predominant reactant on the 30S subunit. Using antibodies raised against the m~A moiety of puromycin, and 30S subunits which lack this modification at the 3'-end of their 16S RNA, the cross-linked puromycin was visualized in the upper head and close to the site determined for S 14'' ~'; (Fig. lb). Since puromycin is an analog of the 3'-terminus of aminoacyl-tRNA, the data suggest that the aminoacyl moiety binds proximal to the head of the 30S subunit. Additional support for this view stems from the labelling of proteins $3, $7, S 13 and S 14 by affinity analogs of tRNA modified at the aminoacyl end. In contrast, several lines of evidence place the decoding site, i.e. the region which binds mRNA and the tRNA anticodon, on the platform and upper body. First, the polypyrimidine sequence believed to base-pair with a preinitiation sequence in mRNA (Shine-Dalgarno interaction) lies very near the 3'-end of the 16S RNA. In addition, the wobble base of the tRNA anticodon can be cross-linked to a cytidine residue at position 1400 ofE. coli 16S RNA ~7, which may lie close to the 3'-end by virtue of the RNA secondary structure (Fig. 3). Finally, the Poustchino group TM have attached haptens to the 5'- and 3'-ends of a 40 to 70nucleotide fragment of polyuridylic acid (ribosomes protect about 30 nucleotides of

361

(6

( 7,9 ,13,19 )

11

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i ~i i ~~iiI

5' El

rnRNA

3'END

Fig. 3. Schematic diagram orE. coli 16S RNA. Black dots are placed every 2 O0 nucleotides from the 5' end. Roman numerals indicate R N A domains; numbers represent 30S proteins which protect regions (outlined) from nuclease digestion or cross-link to sites (indicated by arrows) upon UV irradiation, m R N A Shine-Dalgarno preinitiation sequence; tRNA - site which cross-links to tRNA anticodon. Secondary structure proposed by Noller and Woese (Ref. 15 and personal communication).

mRNA against ribonuclease digestion) and have located a coincident entry and exit site on the exterior surface of the 30S subunit, adjacent to the decoding site, which suggests that the mRNA forms a loop structure during translation. The Berlin group also report preliminary evidence for haptenated poly-4-thiouridylic acid lying in the same general region~. 50 S subunit Less progress has been made with the 50S subunit proteins. No distances between centres of mass have been determined by the neutron scattering method and few protein sites appear on the latest modelsG.19. Our consensus assignments are, therefore, inevitably more subjective than those for the 30S subunit. Account has been taken of the amount and quality of the published IEM data and whether the placement of two

or more proteins as neighbours [for example LI8 and L25, or L10, L l l and (L7/12h] is supported by strong biochemical evidence. By far the best characterized of the proteins is L7/I 2, which exists on the subunit as two dimers. Boublik et al. and Strycharzetal. demonstrated that they lie in a 'stalk-like' projection (Fig. 4 and see Ref. 20). More recent work, by M611er and colleagues, has demonstrated that the 'stalk' can be generated by one protein dimer per ribosome, and they provide evidence that the other dimer, which has a different binding affinity for the ribosome, may fold into the body of the subunit on the interface side 2°. Protein L10 which interacts with L7/12 has been placed at the base of the stalk (Fig. 4). LI 1, which is related to L10 by both structural and functional criteria, is located adjacent to LIO in the revised Berlin modelG. This group of proteins is known to

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the 50S subunit on its exterior side, away subunits has been proposed by Boublik et from the subunit interface. They proposed aL 32 Moreover, while chemical crossthat there may be a tunnel through the body linking of protein pairs located at the subof the 50S subunit through which all nas- unit interface, with short reagents 33, has cent proteins are threaded in an extended provided some support for the models in form. The site is located some 150 (-+30) A Fig. 5 (e.g. S l l - L 1 and S13-L18 are from the peptidyl transferase centre (Fig. cross-linked), the majority of the detected pairings are inconsistent with these models 4). The 3'-end of the 23S RNA was located (e.g. S4-L1, S8-L1 and S10-L1 contain on the back of the 50S subunit by the Pous- proteins located on the exterior surface of tchino group2g using the same technique as the 30S subunit and distant from protein for the 3'-ends of the 16S and 5S RNAs, L1). If the cross-linking data are valid, and this location was confirmed by the Ber- these discrepancies may reflect either errors lin group=4; since the 3'- and 5'-terminal in the subunit alignment, or that some prosequences are base-paired 3°, the latter must teins extend through the subunit but are also share this location. The protein-RNA inaccessible to antibodies on the subunit relationships are less well defined for the interface. 50S subunit. The 23S RNA contains six large structural domains; (L7/12h-L10, Evolution of the IEM model A striking aspect of the early work was L11 and EF-G have attachment sites within domain II (nucleotides 579-1261 ) whereas the finding that many proteins had widely the 5S RNA-protein complex and L1 are separated antigenic determinants on the ribosomal surface; the most extreme examassociated with domain V (2043-2625)3°. ples, from the Berlin group, were protein S15 (tool. wt 10 001) and S18 (tool. wt 70S ribosome Using both single and double antibody 8 896) with multiple sites about 250/~ and labelling of 70S ribosomes, Lake has local- 200 A apart, respectively. It was proposed ized antigenic sites on the intact particle and that these, and other proteins, exhibited determined the relative orientation of the highly extended conformations within the two subunits al. With similar double- ribosome. This conclusion received some labelling experiments, the Berlin group has support from solution studies on proteins, recently achieved similar results °. The two isolated in 6 M urea, which yielded high models are depicted schematically in Fig. estimates for the gyration radii; however, 5. An earlier model from the Berlin group, many of the same proteins, when subjected in which the 30S subunit lies with its long to limited proteolysis, also produced large axis on a line between the stalk and small resistant fragments. More recently, proprotuberance of the 50S subunit, has thus teins prepared under mildly denaturing been superseded. Although the current conditions have yielded lower gyration models are consistent with certain aspects radii estimates (with the possible exception of ribosome function in that, for example, of protein $4), but similar protease fragthey bring together the puromycin sites on ments. It seems probable, therefore, that a the 50S and 30S subunits into a single pep- fraction (and possibly a large one) of the tidyl transferase centre, there are still major proteins used in the earlier solution studies discrepancies between these models and was denatured. The antigenic sites that other data. An alternative alignment of the were detected in the earlier Berlin and UCLA models have been concisely comCENTRAL pared by Gaffney and Craven 34 who b. a. p/PROTUBERANCE emphasized the extensive differences between the two models (this article also STALK ~ covers the early literature). As the maps have evolved the multiple determinants for single proteins have been eliminated leaving one and often no sites. To some degree, the multiple sites can be attributed to cross-contamination of the antibody preparations, a problem which underscores the difficulty in obtaining highly purified ribosomal proteins by conventional methods. This view is supported by the higher level of agreement obtained in localizing RNA determinants where there is a unique site. However, the frequency of multiple sites was highest in the Berlin EXTERIOR INTERFACE Fig. 4. Consensus model of 50S subunit. (a) exterior view; (b) interface view. E = exit site of nascent poly- model and, here, interpretive problems may also have contributed, owing to their peptide; for explanation of other symbols see legend to Fig. 1. Subunit shape after Lake and co-workers 1".

be involved in EF-G-dependent GTP hydrolysis and Vasiliev and colleagues have localized antibodies against EF-G in this neighbourhood 21 (see Fig. 4b). There is also preliminary evidence that immunoglobulins raised against thiostrepton, which inhibits EF-G binding to the 50S subunit, also attach in this region22. The validity of these results is strengthened by the observation that LI1, EF-G and thiostrepton all attach to the same small RNA region. The 3'-end of the 5S RNA was mapped using the same immunochemical technique as was developed for localizing the 3'-end of the 16S RNA. The Poustchino group 2s showed that it occurred on the central protuberance (confLrmed by the Berlin group 24) and they predicted that protein L25, which binds close to the 3'-end of 5S RNA, would occur in the same region. Recently, both of the strong 5S RNA binding proteins L25 and L18 have, indeed, been placed in this locality by the Berlin group ° (Fig. 4). Adjacent to the central protuberance, several sites have been localized which lie in, or close to, the peptidyl transferase. Protein L27, which has been chemically crosslinked to the modified 3'-end of aminoacyl tRNA, bound in either the peptidyl or the aminoacyl site, lies on one side of the central protuberance, and puromycin, which binds to the peptidyl transferase centre, has been chemically cross-linked primarily to protein L232s and is localized adjacent to the central protuberance 25'2e. Protein L1, the first protein to be localized, albeit on a pseudosymmetrical model, lies on the small protuberance as depicted in Fig. 4 t',2~, adjacent to the peptidyl transferase centre. Bernabeu and Lake 28, using a mutant of E. c o l i that overproduces fl-galactosidase, established that the nascent polypeptide (the C-terminal 30--40 amino acids) leaves

I

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363

TIBS - O c t o b e r 1983 O.

b.

Fig. 5. Relative orientation of subunits in the 70S ribosome. Site locations of four proteins are shown for comparison. (a) after LakeS'; (b) after St6ffler and co-workers6.

earlier use of pseudosymmetrical models for both subunits; their double sites for $3 and S10, for example, were mirror-image duplications of the single sites for each protein in the UCLA model. A few control experiments have been introduced in order to establish the specificity of the localized IgG attachment sites. An assessment of the total yield of IgG-linked subunit 'dimers' is corroborative when the figure is high, although low yields might have more to do with steric factors imposed by the requisite orientation of the subunits. To measure the specificity more directly, Lake and co-workers 4 have performed two types of reconstitution experiments. In one method they omit a single protein from the 30S reconstitution mixture and demonstrate a concomitant loss of'dimer' yield with lgG raised against that protein. In a comparable control experiment the Berlin group have employed ribosomes isolated from mutants which are deficient in a single protein 27. The second method used by the UCLA group is analogous, except that the omitted protein is replaced with the equivalent protein from B. s t e a r o t h e r m o p h i l u s . While the reduction in dimer yield is generally dramatic, these control experiments are often difficult to interpret in view of the decreased functional activity and potentially altered conformation of the chimeric 30S subunits. Conclusion The purpose of this review is to produce a minimal structural model of the ribosome that can be used with some confidence in future research. It can be added to (and revised) as more data become available. Our main criterion for reliability, namely that at least two groups should agree, is obviously not foolproof, especially when

one considers the large number of changes that have occurred in the IEM data over the past few years. However, the current awareness of the technical difficulties, particularly in applying the immune electron microscopy method to ribosomes, has instilled considerable caution in purifying immunoglobulins and in designing experiments such that future results are likely to be more accurate. Acknowledgements We thank all those colleagues who sent manuscripts prior to publication or who critically read this review. The review was made possible by a NATO travel grant shared by R. A. Garter and Prof. H. Noller, J. B. Prince is supported by the US National Institutes of Health postdoctoral fellowship GM-08504. R. R. Gutell is supported by the US National Institutes of Health grant GM- 17129 (awarded to H. F. Noller). R. A. Garrett received grants from the Danish Science Research Council. References 1 st6ffler, G., Hasenbank, R., Liitgehaus, M., Maschler, R , Morrison, C. A., Zeichhardt, H. and Garrett, R. A. (1973)Mol. Gen. Genet. 127, 89-110 2 Ramakfislman, V. R., Yabaki, S., Sillers, I.-Y., Schindler, D. G., Engelman, D. M. and Moore, P. B. (1981)J. Mol. BioL 153,739-760 3 Sillers, I.-Y. and Moore, P. B. (1981) J. Mol. Biol. 153,761-780 4 K~an, L., Winkelmann, D. A. and Lake, J. A. (1981)J. Mol. Biol. 145, 193-214 5 Winkelmann, D. A., Kahan, L. and Lake, J. A. (1982) Proc. Natl Acad. Sci. USA 79, 5184-5188 6 Kastner, B., Noah, M., St6ffler-Meilicke, M. and St/~ffler, G. (1983)Proc. 10th Int. Congr. Elect. Micros. 3, 99-106 7 Shatsky, I. N., Mochalova, L. V., Kojouharova, H. S., Bogdanov, A. A. and Vasiliev, V. D. (1979)J. Mol. Biol. 133,501-515

8 Mochalova, L. V., Shatsky, I. N., Bogdanov, A. A. and Vasiliev, V. D. (1982)J. Mol. Biol. 159, 637-650 9 Korn, A. P., Spitnik-Elson, P. and Elson, D. (1982)J. BioL Chem. 257, 7155-7160 10 Spiess, E. (1979)Eur. J. Cell. Biol. 19, 120-130 11 Olson, H. M. and Glitz, D. G. (1979) Proc. Natl Acad. Sci. USA 76, 3769-3773 12 LUlmrman, R., StSffler-Meilicke, M. and StSffler, G. (1981) Mol. Gen. Genet. 182, 369-376 13 Politz, S. M. and Glitz, D. G. (1977) Proc. Natl Acad. Sei. USA 74, 1468-1472 14 Trempe, M. R., Ohgi, K. and Glitz, D. G. (1982) J. Biol. Chem. 257, 9822-9829 15 Noller, H. F. and Woese, C. R. (1981)Science 212, 403-411 16 Olson, H. M., Grant, P. G., Glitz, D. G. and Cooperman, B. S. (1980)Proc. Natl Acad. Sci. USA 77, 890--894 17 Prince, J. B., Taylor, B. H., Thurlow, D. L., Ofengand, J. and Zimmermann, R. A. (1982) Proc. Natl Acad. Sci. USA 79, 5450-5454 18 Evstafieva, A. G., Shatsky, I. N., Bogdanov, A. A., Semenkov, Y. P. and Vasiliev, V. D. (1983) E M B O J. 2,799--804 19 Lake, J. A. and Strycharz, W. A. (1981)J. Mol. BIOL 153,979-992 20 M611er, W., Schrier, P. I., Maassen, J. A., Zantema, A., Sehop, E., Reinalda, H., Cren~rs, A. F. M. and Mellema, J. E. (1983)J. Mol. Biol. 163, 553-573 21 Girshovich, A. S. J., Kurtskhalia, T. V., Ovehinnikov, T. U. A. and Vasiliev, V. D. (1981 ) FEBS Lett. 130, 54-59 22 St6ffler, G., Bald, R., Liihnnann, R., Tischendorf, G. and StSffler-Meilicke, M. (1980) 7th Europ. Congr. in Elect. Micros., Leiden, Vol. 2, p. 566-567 23 Shatsky, I. N., Evstafieva, A. G., Bystrova, T. F., Bogdanov, A. A. and Vasiliev, V. D. (1980) FEBS Lett. 121,97-100 24 St/Sffler-Meilicke, M., StOffler, G., Odom, O. W., Zinn, A., Kramer, G. and Hardesty, B. (1981) Proc. Natl Acad. Sci. USA 78, 5538-5542 25 Olson, H. M., Grant, P. G., Cooperman, B. S. and Glitz, D. G. (1982)J. Biol. Chem. 257, 2649-2656 26 Liihrmann, R., Bald, R., St6ffler-Meilicke, M. and Stfffler, G. (1981) Proc. Natl Acad. Sci. USA 78, 7276-7280 27 Dabbs, E. R., Ehrlich, R., Hasenbank, R., Schroeter, B.-H., St/~ffler-Meilicke, M. and St6ffler, G. (1981)J. Mol. Biol. 149,553-578 28 Bernabeu, C. and Lake, J. A. (1982) Proc. Natl Acad. Sci. USA 79, 3111-3115 29 Shatsky, I. N., Evstafieva, A. G., Bystrova, T. F., Bogdanov, A. A. and Vasiliev, V. D. (1980) FEBS Lett. 122, 251-255 30 NoUer, H. F., Kop, J., Wbeaton, V., Brosius, J., Gutell, R. R., Kopylov, A. M., Dohme, F., Herr, W., Staid, D. A., Gupta, R. and Woese, C. R. (1981)Nuc/. Acids Res. 9, 6167-6189 31 Lake, J. A. (1982)J. Mol. Biol. 161, 89-106 32 Boublik, M., Hellmann, W. and Kleinschmidt, A. K. (1977) Cytobiology 14, 293-300 33 Cover, J., Lambert, J. M., Norman, C. M. and Traut, R. R. (1981) Biochem. 20, 2843-2852 34 Gaffney, P. T. and Craven, G. R. (1980) in Ribosomes (Chambliss, G., et al., eds), pp. 237-253, University Park Press