THE J O U R N A L OF BIOLOGICAL C H E M I S T R Y e 1984 by The American Society of Biological Chemists, lnc.

Vol. 259, No. 24, Issue of December 25, pp. 15257--15263, 1984 Prlnted in U.S A.

Mobile Domains in Ribosomes Revealed by Proton Nuclear Magnetic Resonance*

(Received for publication, July 17, 1984)

Cynthia A. Cowgill$, Brenda G. Nichols, James W. Kenny& Peter Butler?, E. Morton Bradbury, and Robert R. Traut I1 From the Department of Biological Chemistry, School of Medicine, University of California, Davis, California 95616

Ribosomes and subunits from eukaryotic and pro- karyotic sources were studied by high-resolution pro- ton magnetic-resonance spectroscopy. If all ribosomal components are firmly bound within the particle, then only broad spectra would be expected. However, rela- tively sharp resonances were found both in ribosomal subunits and in 70 or 80 S ribosomes. The regions of these mobile protein domains have been partially as- signed in Escherichia coli ribosomes. Large and small ribosomal subunits were treated to remove selectively proteins L7/12 and SI, respectively. Sharp proton magnetic resonance spectra were not observed for the stripped large subunit showing that proteins L7/12 comprise the flexible protein region and that there is little other flexibility in the stripped subunit. Complete removal of S1 from the small subunit greatly reduced but did not abolish the sharp protein resonance peaks, indicating that protein SI contains a substantial flex- ible component but that other flexible components re- main in the stripped small subunit. Evidence for gen- erality of these features of ribosome organization is provided by similar studies on ribosomes from eukar- yotic sources.

The ribosome is a complex structure which interacts with many ligands to synthesize protein. How the ribosome works at the molecular level is not understood, although in Esche- richia coli the sequences of all its components are known. The protein topography of E. coli 30 S ribosomal subunits has been studied by neutron scattering, immune electron micros- copy, and protein-protein cross-linking. Some protein posi- tions for the 50 S subunit have also been established. These methods provide a relatively static map of ribosomal constit- uents. However, the ribosome during its functional cycle is clearly a dynamic structure. For example, a central functional step, translocation, involves movement of the ribosome rela- tive to messenger and peptidyl tRNAs in which mobile ribo- somal constituents or domains may be involved (for reviews, see Refs. 1-4). The characterization of conformational

* This work was supported by grants to R. R. T. from the United States Public Health Service (GM 17924), the American Cancer Society (NP252), and the University of California. We thank Drs. A. Wahba, J. Lee, and S. Michel for providing ribosome from Artemia yeast and rat liver, respectively. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address: Cutter Laboratories, Erneryville, CA 94701, § Present address: Calgene Incorporated, Davis, CA 95616. B Present address: Microbiolgy Unit, Department of Mirobiology,

1) To whom correspondence should be addressed. University of Oxford, Oxford OX1 3QU, United Kingdom.

changes that take place during protein synthesis is essential to understanding the mechanism.

The results of both functional and structural studies suggest proteins L7/12 as likely candidates for movement within the ribosome. These proteins, which are present on the ribosome in 4 copies, are necessary for translocation, GTP hydrolysis, and the binding of several factors (1). The position of proteins L7/12 has been located by electron microscopy as a stalk extending away from the body of the 50 S subunit to which it is anchored (5). It has not been determined whether the L7/ 12 stalk is stationary or whether it can rotate or otherwise move relative to the ribosome.

Protein-protein cross-linking studies performed in this lab- oratory have established several proteins which cross-link with L7/12 (6). One significant cross-linked protein is L5 which by immune electron microscopy is located in the central protuberance.’ We have postulated elsewhere that one or more copies of proteins L7/12, while anchored at one end, may move laterally from its position in the stalk across the body of the subunit to a position within cross-linking distance of protein L5 (6).

We undertook this proton NMR study to investigate the presence of mobile domains in ribosomes and the possibility that proteins L7/12 may be involved. The ribosome is so large that if it were rigid no useful data could be derived from its proton NMR spectrum. However, if there were groups within the ribosome with modalities additional to the tumbling of the ribosome in solution, then they would generate a sharp spectrum. We have observed such sharp spectra and have sought their source in E. coli ribosomes. We establish here that the mobile elements of the large ribosomal subunit are proteins L7/12. A similar mechanism is observed for eukar- yotic large subunits, a result which suggests mobility of the homologous P proteins. Furthermore, the small subunit also has a highly mobile region made up partly of protein SI, and both of these flexible domains appear to be a general feature of ribosomes from many sources. During the course of this study a similar finding was reported for the large E. coli subunits (8).

EXPERIMENTAL PROCEDURES

Ribosomes and ribosomal subunits from E. coli, yeast, Artemia salina, rabbit reticulocytes, and rat liver were prepared by standard procedures (9-13). E. coli cells were ground with alumina. Eukaryotic cells were ruptured and centrifuged at low speed to remove cell debris. Ribosomes and polysomes present in the supernatant were then pelleted at high speed through a sucrose cushion. Ribosomal subunits were prepared by resuspending the pelleted ribosomes in the appro- priate low-magnesium buffer and isolating the subunits by gradient centrifugation in solution containing sucrose. Yeast ribosomal sub-

’ A. Sommer, H. Olson, R. R. Traut, and D. G. Glitz, unpublished results.

15257

15258 Mobile Domains in Ribosomes Revealed by Proton NMR units and one E. coli ribosomal subunit preparation were isolated on gradients containing glycerol.

It was necessary to determine buffer conditions appropriate for the NMR experiments to replace the Tris buffers normally used. Accord- ingly, samples containing 2-20 mg/ml of each ribosomal species were dialyzed for 3 days in deuterated phosphate buffer, after which their integrity was determined by sucrose gradient analysis prior to NMR analysis. The phosphate buffer conditions for each species are stated in the figure legends.

Fig. 1 shows the sucrose gradient profiles of the ribosomal subunits from Artemiu which were obtained on samples immediately prior to NMR analyses. The subunits are stable in the phosphate buffer used. Similar results were obtained for ribosomal particles from the other species (results not shown). Protein S1 was removed from the 30 S subunit of E. coli by successive centrifugation through a 20% glycerol gradient (14). Proteins L7/L12 were removed from E. coli 50 S subunits and ribosomes by their selective extraction in 0.5 M NH&I and 50% ethanol (15). Figs. 2 and 3 show by sucrose gradient analysis the integrity of E. coli 50 and 30 S cores, respectively, from which L7/L12 and S1 have been removed and which have been dialyzed against deuterated phosphate buffers. Fig. 4 shows the analysis by gel electrophoresis of the core proteins and of the proteins extracted. Proteins L7/L12 and S1 are the only proteins extracted from 50 and 30 S subunits.

All spectra were recorded on a Nicolet NT-360 MHz spectrometer except for those of the rabbit reticulocyte which were recorded on a Nicolet NT-500 MHz spectrometer. Spectra were recorded at a tem- perature of 19 "C with a sweep width of 6000 Hz. The experiments employed a low-power preirradiation pulse of 0.56 s to suppress the residual water in the samples. All spectra were recorded from 5-mm sample tubes. A 45 "C flip angle was applied. The repetition rate was

- Sedimentotion FIG. 1. Sucrose gradient analysis of Artemia ribosomes and

subunits immediately prior to NMR analysis. Ribosomes were dialyzed twice in aqueous phosphate buffer, 10 mM KCl, 20 mM sodium phosphate, pH 7.2, 0.75 mM MgC12, and three times in deuterated phosphate buffer. Subunit buffer contained 0.02 mM MgCl,. Ribosomes and subunits (0.1 mg) were applied to 7-25% sucrose gradients and centrifuged for 90 min in an SW 56 rotor at 54,000 rpm and 19 "C. A, 40 S subunits; B, 60 S subunits; C, 80 S ribosomes.

06 1 A I

Sedirnentotton -+

FIG. 2. Sucrose gradient analysis of E. coli 50 S subunits and cores which are minus proteins L7&12. Subunits and cores were prepared for NMR and sucrose gradient analysis as described in Fig. 1 using phosphate buffer, 100 mM KC1, 20 mM sodium phosphate, pH 7.2, 2 mM MgCl,. A , 50 S subunits; B, 30 S cores.

06 -

0 04 - a B

0 2 A B

06 -

0 04 - a B

0 2 A B

Sedirnentotion -+

FIG. 3. Sucrose gradient analysis of E. coli 30 S subunits and cores which are minus protein S1. Samples were prepared as in Fig. 2. A, 30 S and subunits; B, 30 S cores.

approximately 1 s. The number of scans collected depended on the sample concentration and ranged from 2,000-10,000.

RESULTS

The contribution of proteins L7/L12 to the NMR spectrum of E. coli 50 S ribosomal subunits was investigated. The spectra of pure L7/L12, intact 50 S subunits, cores deficient in L7/L12, and reconstituted 50 S subunits are shown in Fig. 5. Proteins L7/L12 show a number of sharp resonance lines in the regions from 0 to 3.4 ppm (Fig. 5A) and 6.5 to 7.5 ppm (Fig. 6D). These sharp lines are evident in the spectrum of the intact 50 S subunit (Figs. 5B and 6E). By contrast, the core particles from which L7/L12 has been specifically and completely removed lack any sharp resonance lines in these regions (Fig. 5C and 6F). The sharp resonances reappear in the reconstituted 50 S particles formed by readdition of L7/ L12 to the cores (Fig. 50).

Similar experiments were performed with intact 70 S ribo- somes and 70 S ribosomes specifically lacking L7/L12. The results are shown in Fig. 7. A comparison of the spectra of 70 S (Fig. 7A) and 70 S minus L7/L12 (Fig. 7B) shows a dimi- nution of sharp resonances in the core, but not a complete absence. The resonances are mostly restored by reconstituting the cores with L7/L12 (Fig. 7C).

Experiments were performed with E. coli 30 S subunits to assess the contribution of protein S1 to the NMR spectrum. Figs. 6 and 8 compare the spectra for pure S1, intact 30 S particles, and 30 S core particles completely lacking S1. Pro- tein S1 shows sharp resonance lines in the regions from 0 to 3.4 ppm and 6.5 to 7.5 ppm (Figs. 6A and 8A). These sharp lines are also evident in the spectrum obtained with intact 30 S subunits. The core particle lacking S1 shows a significant loss of sharp resonances as compared with the intact 30 S subunit (Figs. 6C and 8C). By contrast to the 50 S core, the 30 S core retains some sharp lines.

The spectra for 70 S ribosomes were determined at two magnesium concentrations. At the high concentration, 20 mM, the particles exist as 70 S couples. At the low concentration, 2 mM, the particles exist as 30 and 50 S subunits as demon- strated by sucrose gradient centrifugation (not shown). The sharp spectra characteristic of the dissociated subunits, shown above to originate from L7/L12 in the 50 S subunit and in large part S1 in the 30 S subunit, are also present in the original 70 S couples and in the reassociated couples (Fig. 9, A, B , and C).

The spectra of ribosomal subunits prepared by centrifuga- tion in solutions containing both 0.5 M NH&l and sucrose show characteristic sharp resonances around 3.6 to 4 (Figs. B and C and 8, B and C). They are not seen in the spectra of 70 S ribosomes prepared in the absence of sucrose (Figs. 7 and 9) nor are they seen when glycerol is substituted for sucrose in the gradient centrifugation to prepare subunits (Fig. 10).

Mobile Domains in Ribosomes Revealed by Proton N M R 15259

a. "

FIG. 4. Analysis by gel electro- phoresis of the core proteins and of the proteins extracted. a, 50 S subunit proteins analyzed by two-dimensional acid urea gel electrophoresis (29); b, 50 S core proteins; c, extracted proteins L7/ L12 analyzed by SDS-gel electrophoresis (7): d, extracted protein S1; e, 30 S core proteins.

50s PROTEINS CORE PROTEINS 7/12 SI 30s -S I

i, - l ~ ' l l l ~ l l l l ~ ' l l ' ~ l l l l ~ ' " '

4 3 2 I 0

PPm FIG. 5. Spectra of pure proteins L7/L12 ( A ) , intact 50 S

subunits ( B ) , cores deficient in L7/L12 (0, and reconstituted 50 S subunits ( D ) .

Purified proteins L7/L12 and S1 do not show these lines (Figs. 5A and 8A).

Spectra were obtained for eukaryotic polyribosomes, 80 S

,' D X"" "" 1

. - - - -. ,""

. .

FIG. 6. Spectra of the aromatic region of E. coli protein S 1 (A) , 30 S subunits (B) , 30 S cores (C), proteins L7/L12 (D) , 50 S subunits ( E ) , and 50 S cores (F).

ribosomes, and 60 and 40 S ribosomal subunits from rabbit reticulocytes and for ribosomes and subunits from rat liver, Artemia, and yeast. The 40 S spectra (Figs. 11, A, B and C and 13C) and the 60 S spectra (Figs. 12, A, B, and C and 13 C) of four eukaryotic species all contain sharp resonances in the 0- to 3.4-ppm region as do E. coli subunits. A comparison of the spectra of rabbit reticulocyte subunits with 80 S and polysomes (Fig. 13, A, B, C, D) shows the maintenance of resonances through the polysome level of organization.

DISCUSSION

Sharp resonances found by proton NMR analysis of intact ribosomes and ribosomal subunits indicate the presence of mobile protein domains in the ribosome. Protons present in residues tightly bound to a structure as massive as the ribo- some would not give rise to sharp resonances but rather to an extremely broad envelope of resonances. Identification of some mobile ribosomal elements was accomplished by selec- tive extraction of two ribosomal proteins and spectral analysis of the remaining particles. Multienzyme complexes, such as the pyruvate dehydrogenase complex and its "partial" com- plexes have been studied by this qualitative approach. By omitting successive components of the complex and studying their spectra, the mobile region was located in only one of the three component enzymes (16).

15260 Mobile Domains in Ribosomes Revealed by Proton NMR

A

. . , " , , , " " , , , , Y 3 2 1 0 -1

PPm FIG. 7. Spectra of E. coli 70 S ribosomes (A), 70 S cores

which leek L7/L12 (B), and 70 S particles reconstituted with L?/L12 (C). These samples were prepared in phosphate buffer, 100 mM KCl, 20 mM sodium phosphate, pH 7.2, 20 m M MgC1,.

The 50 S core particles lacking only the four copies of L7/ L12 show no sharp resonance lines (Fig. 5) . When the proteins are used to reconstitute intact 50 S subunits, the repurified intact particles again show the sharp resonances. We interpret the results to show that L7/L12 retain mobility when an- chored as part of the 50 S structure. We cannot exclude that the removal of L?/L12 would lead to the tighter attachment of an otherwise mobile domain. The mobility of L7/L12 implied by the NMR results is consistent with other data: a fluorescent dye attached to the COOH termini of L7/L12 was very accessible to solvent and the region has a high degree of rotational mobility (17); a spin label attached near the NH2 terminus of L7/L12 is also mobile (18).

Experiments were performed to assess the effect of the association of 30 S subunits to 50 S subunits on the spectrum (Fig. 9). There was no significant difference between the spectrum obtained at 2 mM M e , conditions in which the subunits were completely dissociated as shown by sucrose gradient centrifugation, and at 20 mM Mg', conditions in which the subunits were completely associated. While both

v u v...,.,,,,,.,.,k 4 3 2 1 0 - I

PPm FIG. 8. Spectra of E. coli protein S1 (A) , 30 S subunits (B),

and 30 S cores which lack S1 (C).

subunits contribute to the sharp resonances characteristic of 70 S ribosomes, the results show that the sharp resonances are independent of M$* concentration and that the mobile domains identified in subunits retain comparable mobility upon subunit reassociation.

A more detailed comparison of the spectra of pure L7/L12 and the 50 S subunit or reconstituted 50 S subunit (Fig. 5) provides some information concerning residues of L7/L12 involved in binding. The 0.8- and 1.4-ppm resonances gener- ated by apolar amino acids are broadened when L7/L12 is integrated in the particle and the 1.3-ppm resonance, also generated by apolar amino acids, nearly disappears. These changes suggest an involvement of the corresponding residues in binding. Proteins L7/L12 bind, via LlO, to the 50 S subunit through the NH2-terminal part of the molecule (19, 20). The shortest NH,-terminal fragment shown to interact with the subunit is 1-55. This fragment is rich in apolar amino acid residues (Alal2, Gly,, Ile4, Leu, Pro, Ser4, Thrl, Val8) when compared with the COOH-terminal fragment (21). The NMR results are consistent with a primary involvement of this

Mobile Domains in Ribosomes Revealed by Proton NMR 15261

PPm FIG. 9. Spectra of E. coli 70 S ribosomes prepared at two

magnesium concentrations. A , at 20 mM MgCL; B, at 2 mM; c at 20 mM again.

segment of the molecule in binding to the 50 S. The peaks in Fig. 4a between 3.5-4.4 ppm, representing proline, serine, glycine, and isoleucine, are masked in Fig. 4, b-d, since these particles were prepared by procedures employing 0.5 M salt and sucrose. The 50 S subunits prepared in glycerol (Fig. 8) reveal this region clearly and show the near absence of the sharp resonances characteristic of the pure protein. The res- onances generated by mobile aromatic residues in the protein are also broadened in the subunit (Fig. 6) suggesting their involvement in the binding site. The only aromatic amino acid residues present in L7/L12 are Phe 30 and Phe 54 both in the NH2-terminal segment (22).

Proteins L7/L12 were also selectively removed from the 70 S ribosome. The spectrum of these 70 S cores shows a dimi- nution in peak intensities when compared to the spectrum for intact ribosomes (Fig. 7, A and B) . The reduced intensity is found mainly in two regions, the peaks at 0.8 ppm and 3.5- 4.4 ppm. The sharp resonances remaining are due to flexibility in the 30 S subunit, as we will discuss. The 0.8-ppm peak reappears when L7/L12 are rebound to the 70 S cores, but the resonances in the 3.5-4.4 ppm region do not (Fig. 7C).

The spectrum for intact 30 S ribosomal subunits was com- pared to that for 30 S core particles from which S1 was removed (Fig. 8, B and C ) . The sharp peaks characteristic of

L

n

Y 3 2 1 0 - 1

PPm FIG. 10. Spectrum of E. coli 50 S subunits prepared by

glycerol gradient centrifugation.

A

PPm FIG. 11. Spectra of 40 S subunits from yeast (A) , Arfemia

(B), and rat liver (C). Samples were prepared in phosphate buffer, 10 mM KC1,20 mM sodium phosphate, pH 7.2,O.OZ mM MgC1,.

15262 Mobile Domains in Ribosomes Revealed by Proton N M R

A

e ~ ” - ~ - - - ; ~ , , , , , ~~~~~ . . , , , , , I

7 6 5 Y 9 2 1 0

PPm FIG. 12. Spectra of 60 S subunits from yeast (A) , Artemiu

(B), and rat liver (0.

intact 30 S subunits are reduced in intensity in the core particles. Protein S1 clearly contributes substantially to the pattern of sharp peaks and must have mobile elements when integrated into the particle. In contrast to the 50 S core, which lacks evidence of mobile elements other than L7/L12, the 30 S core retains sharp peaks indicating the presence of mobile elements in addition to protein S1. Sucrose gradient analysis of 30 S cores (Fig. 3) shows that upon removal of S1, the integrity of the core particle is compromised. This may lead to the exposure of new mobile groups. A comparison of the spectra of S1 alone (Fig. &I) and bound as part of 30 S shows evidence that the binding site of SI must contain a large proportion of apolar amino acids (e.g. valine, leucine, and isoleucine, but probably not much alanine). Protein S1, like L7/L12, binds to the ribosome via its NH, terminus which is highly apolar (46%) and has two domains connected by a hinge region (23). Protein S1 is rich in aromatic residues, 66% of which are found in the COOH-terminal half of the molecule (23). The spectrum in the aromatic region shows, however, that most of the mobile aromatic amino acids in the free protein are immobilized when S1 is bound in 30 S subunits (Fig. 6, A, B, C).

The standard preparation of ribosomal subunits using 0.5 M salt and sucrose gradient centrifugation results in particles whose spectra show four strong sharp resonances around 4 ppm. These resonances remain even after extensive dialysis

A

B

C

1 1 1 0 9 8 7 6 S y 3 2 1

PPm FIG. 13. Spectra of rabbit reticulocyte polysomes (A) , 80 S

ribosomes (B), 60 S subunits (C), and 40 S subunits (0). Samples were prepared in phosphate buffer, 10 mM KCI, 20 mM sodium phosphate, pH 7.2, 0.5 mM MgClz.

in either low or high salt. This region of the spectrum has been deleted in presentations of some authors (8) and is clearly visible in the spectra of others (24). Ribosomes exposed to low salt (up to 150 mM) and sucrose show no resonances in this region (data not shown). Ribosomal subunits prepared using high salt and glycerol gradients also do not contain these resonances (Figs. 7, 9, llA, and 12A). Proteins which are extracted from ribosomes lack these resonances (Figs. 5A and 8A). Phenol-extracted RNA from subunits prepared in high salt and sucrose still contain the resonances in the 4- ppm region although they no longer contain protein reso- nances (data not shown). It appears that in high salt the ribosome conformation “breathes” allowing the sucrose to enter where it is then irreversibly trapped by the RNA moiety. There is other evidence that sugars irreversibly affect RNA molecules. Both sucrose and glycerol will stimulate the deac- ylation of aminoacyl-tRNA (25).

The presence of mobile domains in eukaryotic ribosomes was investigated and spectra have been obtained for eukar- yotic ribosomes and subunits from yeast, Artenia, rabbit reticulocyte and rat liver. The spectra of ribosomal subunits from eukaryotic sources (Figs. 11, 12, and 13, C and D) show a number of quite narrow peaks which are even sharper than in E. coli ribosome spectra, indicating more mobile domains in the eukaryotic particles.

An examination of the spectra generated by the mobile components of yeast, Artemia, and rat liver 40 S subunits (Fig. 11) shows that the relative contribution of individual amino acids differs from species to species. For example, the yeast mobile component contains little Lys (3.0 ppm) relative to valine, isoleucine, and leucine (0.8 ppm) when compared with rat liver. It also contains a larger amount of freely moving threonine than any species studied. The observed spectral

Mobile Domains in Ribosomes Revealed by Proton NMR 15263

differences among species appear not directly related to “ev- olutionary complexity”; the ratio between the 3.0- and the 0.8-ppm peak in the rabbit reticulocyte spectrum (Fig. 130) is more similar to that of Arteniu than to rat liver. A large portion of the mobile component in the small subunit of E. coli has been identified as protein S1. Although no protein homologous to S1 has been identified in eukaryotic small subunits, the NMR data show the presence of similarly mobile domains.

An analysis of the spectra resulting from the 60 S subunit mobile region leads to a similar conclusion (Fig. 12). The spectra are significantly different among the species examined and suggest dissimilar mobile domains. Proteins in large eukaryotic subunits homologous to L7/L12 in E. coli have been characterized (26) and are designated P proteins. Like L7/L12 they are relatively acidic, form dimers in solution, and can be selectively extracted by high salt plus ethanol (25). Although there is limited structural homology between L7/ L12 and the P proteins, there is functional homology (27,28). We postulate that the mobile component of the eukaryotic large subunit may consist largely of the P proteins.

The aliphatic mobile resonances in spectra of eukaryotic ribosomal subunits are also present in spectra of intact 80 S ribosomes (Fig. 13B). The aromatic region of the 80 S rabbit reticulocyte spectrum is broad with few sharp components. Because the 40 and 60 S subunit spectra have a more promi- nent aromatic region (Fig. 13, C and D), it seems likely that the mobility of the aromatic amino acids is lowered on asso- ciation of the subunits to form the 80 S ribosome. The 40 S subunit spectra (Fig. 11, A and B) of yeast and Artemia show little evidence of mobile aromatic amino acids while their 60 S counterparts (Fig. 12, A and B) both have sharp aromatic resonances (7.4 ppm). Rat liver shows evidence of mobility in the aromatic region in both the 40 and 60 S subunits (Figs. 11C and 12C). Rabbit reticulocyte polysomes, in contrast with ribosomes, again exhibit mobile aromatic groups as did the subunits implicating a unique polysome conformation (Fig. 13A).

The spectrum of the eukaryotic polysomes examined shows significant line broadening and suggests slower movement in the mobile domains, This could be due to the involvement of the mobile constituents in the binding of factors and ligand RNAs. In E. coli protein S1 is necessary for message RNA binding, and proteins L7/L12 are essential for EF-G factor binding.

The presence of mobile protein. elements appears to be a general property of ribosomes from eukaryotes as well as prokaryotes. The relationship of specific mobile ribosomal domains to ribosome function and the degree and orientation of the motions observed are subjects for future investigation.

The NMR peaks for the four eukaryotes examined appear narrower than those for E. coli suggesting a greater degree of flexibility for certain elements. Identification of the mobile proteins in eukaryotic ribosomes remains to be accomplished.

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