Comparative cross-linking study on the 50S ribosomal subunit from Escherichia coli

We have carried out an extensive protein-protein cross-linking study on the 50S ribosomal subunit of Escherichia coli using four different cross-linking reagents of varying length and specificity. For the unambiguous identification of the members of the cross-linked protein complexes, immunoblotting techniques using antisera specific for each individual ribosomal protein have been used, and for each cross-link, the cross-linking yield has been determined. With the smallest cross-linking reagent diepoxybutane (4 A), four cross-links have been identified, namely, L3-L19, L10-L11, L13-L21, and L14-L19. With the sulfhydryl-specific cross-linking reagent o-phenylenedimaleimide (5.2 A) and p-phenylenedimaleimide (12 A), the cross-links L2-L9, L3-L13, L3-L19, L9-L28, L13-L20, L14-L19, L16-L27, L17-L32, and L20-L21 were formed; in addition, the cross-link L23-L29 was exclusively found with the shorter o-phenylenedimaleimide. The cross-links obtained with dithiobis(succinimidyl propionate) (12 A) were L1-L33, L2-L9, L2-L9-L28, L3-L19, L9-L28, L13-L21, L14-L19, L16-L27, L17-L32, L19-L25, L20-L21, and L23-L34. The good agreement of the cross-links obtained with the different cross-linking reagents used in this study demonstrates the reliability of our cross-linking approach. Incorporation of our cross-linking results into the three-dimensional model of the 50S ribosomal subunit derived from immunoelectron microscopy yields the locations for 29 of the 33 proteins within the larger ribosomal subunit.     

Ribosomal protein L7/L12 cross-links to proteins in separate regions of the 50 S ribosomal subunit of Escherichia coli

The 50 S ribosomal subunits from Escherichia coli were modified by reaction with 2-iminothiolane under conditions in which 65 sulfhydryl groups, about 2/protein, were added per subunit. Earlier work showed that protein L7/L12 was modified more extensively than the average but that nearly all 50 S proteins contained sulfhydryl groups. Mild oxidation led to the formation of disulfide protein-protein cross-links. These were fractionated by urea gel electrophoresis and then analyzed by diagonal gel electrophoresis. Cross-linked complexes containing two, three, and possibly four copies of L7/L12 were evident. Cross-links between L7/L12 and other ribosomal proteins were also formed. These proteins were identified as L5, L6, L10, L11, and, in lower yield, L9, L14, and L17. The yields of cross-links to L5, L6, L10, and L11 were comparable to the most abundant cross-links formed. Similar experiments were performed with 70 S ribosomes. Protein L7/L12 in 70 S ribosomes was cross-linked to proteins L6, L10, and L11. The strong L7/L12-L5 cross-link found in 50 S subunits was absent in 70 S ribosomes. No cross-links between 30 S proteins and L7/L12 were observed.     

Identification of neighbouring proteins by cross-linking of intact 70 S ribosomes from Escherichia coli

70 S ribosomes from Escherichia coli have been reacted with the bifunctional reagent 1,4-phenyldiglyoxal under near physiological conditions. As a result of the cross-linking reaction a number of high-molecular-weight protein fractions with altered electrophoretic mobility could be isolated. A new chemical procedure has been introduced to reverse the cross-links between proteins at least partially. The cleavage reaction did not affect the gel electrophoretic mobility of the proteins. Thus a direct identification of cross-linked proteins using one- or two-dimensional gels was made possible. Two protein trimers, S3-S4-S5 and L1-S4-S5, as well as five protein dimers, S3-S4, L6-L7/12, L10-L7/12, S9-L19 and L18-L19 could be identified as close neighbours in the E. coli 70 S ribosome. The protein pairs S9-L19 and L18-L19 had previously not been identified as near neighbours using cross-linking studies.     

Neighboring proteins in rat liver 60 S ribosomal subunits disulfide-linked by hydrogen peroxide oxidation or cross-linked with dithiobis(succinimidyl propionate)

Neighboring proteins in rat liver 60 S ribosomal subunits were investigated by two kinds of cross-linking techniques: treatment of 60 S subunits with 1) hydrogen peroxide, which promotes the formation of protein-protein disulfide linkages and 2) a disulfide-bridged bifunctional reagent dithiobis(succinimidyl propionate). The cross-linked protein complexes formed were separated by two-dimensional polyacrylamide gel electrophoresis in a basic-sodium dodecyl sulfate gel system under nonreducing conditions. Each complex in the gel was labeled with 125I and extracted under reducing conditions. The protein components of the complex were analyzed by two kinds of two-dimensional polyacrylamide gel electrophoresis, followed by autoradiography. Closely neighboring pairs disulfide-linked by hydrogen peroxide were identified as L4-L6, L4-L29, L6-L29, L18a-L29, and L29-L32; more distant pairs cross-linked with dithiobis(succinimidyl propionate) were identified as L3-L5, L3-L24, L3-L37a, L4-L14, L4-L18a, L5-L10, L5-L11, L7/L7a-L27, L7/L7a-L36, L13-L35, and L13a-L14.     

Studies on the binding of the ribosomal protein complex L7/12-L10 and protein L11 to the 5''-one third of 23S RNA: a functional centre of the 50S subunit.

The RNA binding sites of the protein complex of L7/12 dimers and L10, and of protein L11, occur within the 5'-one third of 23S RNA. Binding of the L7/12-L10 protein complex to the 23S RNA is stimulated by protein L11 and vice-versa. This is the second example to be established of mutual stimulation of RNA binding by two ribosomal proteins or protein complexes, and suggests that this may be an important principle governing ribosomal protein-RNA assembly. When the L7/12-L10 complex is bound to the RNA, L10 becomes strongly resistant to trypsin. Since the L7/12 dimer does not bind specifically to the 23S RNA, this suggests that L10 constitutes a major RNA binding site of the protein complex. Only one of the L7/12 dimers is bound strongly in the (L7/12-L10)-23S RNA complex; the other can dissociate with no concurrent loss of L10.     

Protein-protein cross-linking of the 50 S ribosomal subunit of Escherichia coli using 2-iminothiolane. Identification of cross-links by immunoblotting techniques

We have investigated the protein-protein cross-links formed within the 50 S subunit of the Escherichia coli ribosome using 2-iminothiolane as the cross-linking reagent. The members of the cross-links have been identified by immunoblotting from one-dimensional and two-dimensional diagonal sodium dodecyl sulfate-polyacrylamide gels using antisera specific for the individual ribosomal proteins. This method also allowed a quantitation of the yield of cross-linking for each cross-link. A total of 14 cross-links have been identified: L1-L33, L2-L9, L2-L9-L28, L3-L19, L9-L28, L13-L21, L14-L19, L16-L27, L17-L30, L17-L32, L19-L25, L20-L21, L22-L32, and L23-L34. Our results are compared with those of Traut and coworkers (Traut, R. R., Tewari, D. S., Sommer, A., Gavino, G. R., Olson, H. M., and Glitz, D. G. (1986) in Structure, Function and Genetics of Ribosomes (Hardesty, B. and Kramer, G., eds) pp. 286-308, Springer-Verlag, New York). Our cross-linking data allow us to propose the approximate locations of eight proteins of the 50 S ribosomal subunit that so far have not been localized by immunoelectron microscopy and they thus contribute considerably to our knowledge of ribosome structure.     

The large subunit of the mammalian mitochondrial ribosome. Analysis of the complement of ribosomal proteins present

Identification of all the protein components of the large subunit (39 S) of the mammalian mitochondrial ribosome has been achieved by carrying out proteolytic digestions of whole 39 S subunits followed by analysis of the resultant peptides by liquid chromatography and mass spectrometry. Peptide sequence information was used to search the human EST data bases and complete coding sequences were assembled. The human mitochondrial 39 S subunit has 48 distinct proteins. Twenty eight of these are homologs of the Escherichia coli 50 S ribosomal proteins L1, L2, L3, L4, L7/L12, L9, L10, L11, L13, L14, L15, L16, L17, L18, L19, L20, L21, L22, L23, L24, L27, L28, L30, L32, L33, L34, L35, and L36. Almost all of these proteins have homologs in Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae mitochondrial ribosomes. No mitochondrial homologs to prokaryotic ribosomal proteins L5, L6, L25, L29, and L31 could be found either in the peptides obtained or by analysis of the available data bases. The remaining 20 proteins present in the 39 S subunits are specific to mitochondrial ribosomes. Proteins in this group have no apparent homologs in bacterial, chloroplast, archaebacterial, or cytosolic ribosomes. All but two of the proteins has a clear homolog in D. melanogaster while all can be found in the genome of C. elegans. Ten of the 20 mitochondrial specific 39 S proteins have homologs in S. cerevisiae. Homologs of 2 of these new classes of ribosomal proteins could be identified in the Arabidopsis thaliana genome.     

Comparative study between prokaryotes and eukaryotes by chemical iodination of ribosomal proteins.

Escherichia coli and Saccharomyces cerevisiae ribosomal proteins were chemically iodinated with 125I by chloramine T under conditions in which the proteins were denatured. The labelled proteins were subsequently separated by two-dimensional gel electrophoresis with an excess of untreated ribosomal proteins from the same species. The iodination did not change the electrophoretic mobility of the proteins as shown by the pattern of spots in the stained gel slabs and their autoradiography. The 125I radioactivity incorporated in the proteins was estimated by cutting out the gel spots from the two-dimensional electrophoresis gel slabs. The highest content of 125I was found in the ribosomal proteins L2, L11, L13, L20/S12, S4 and S9 from E. coli, and L2/L3, L4/L6/S7, L5, L19/L20, L22/S17, L29/S27, L35/L37 and S14/S15 from S. cerevisiae. Comparisons between the electrophoretic patterns of E. coli and S. cerevisiae ribosomal proteins were carried out by coelectrophoresis of labelled and unlabelled proteins from both species. E. coli ribosomal proteins L5, L11, L20, S2, S3 and S15/S16 were found to overlap with L15, L11/L16, L36/L37, S3, S10 and S33 from S. cerevisiae, respectively. Similar coelectrophoresis of E. coli 125I-labelled proteins with unlabelled rat liver and wheat germ ribosomal proteins showed the former to overlap with proteins L1, L11, L14, L16, L19, L20 and the latter with L2, L5, L6, L15, L17 from E. coli.     

Co-operative response of oligomeric protein receptors coupled to non-co-operative ligand binding.

The biogenesis of 30 S and 50 S ribosomal subunits in exponentially growing Escherichia coli has been studied by following the rate of appearance of pulse-labelled ribosomal proteins on mature subunits. Cells were pulse-labelled for two minutes and for three and a half minutes with radioactive leucine. Ribosomal proteins were extracted and purified by chromatography on carboxymethyl cellulose and analysed by bidimensional gel electrophoresis. All 30 S proteins and most of the 50 S proteins were thus prepared and their radioactivity counted: unequal labelling was obtained. 30 S and 50 S proteins were ordered according to increasing specific radioactivity at both time pulses. The incorporation was greater at three and a half minutes than at two minutes. No major difference in the order at the two labelling times was observed.Only two classes of proteins can be defined in the 30 S and the 50 S subunits, namely early and late proteins. In each class a gradual increase in the radioactivity is apparent from the poorly labelled to the highly labelled proteins. This suggests a definite order of addition.Early 30 S proteins: S17, S16, S15, S19, S18, S8, S4, S20, S10, S6, S9, S12, S7.Late 30 S proteins: S5, S3, S2, S14, S11, S13, S1, S21.Early 50 S proteins: L22, L20, L21, L4, L13, L16, L3, L23, L18, L24, L28, L17, L19, L29, L32, L5, L15, L2, L30, L27.Late 50 S proteins: L25, L11, L7, L12, L1, L9, L8, L10, L33, L14, L6.This order is discussed taking into account the pool size of the proteins measured in the same conditions of cell culture.     

Translation elongation by a hybrid ribosome in which proteins at the GTPase center of the Escherichia coli ribosome are replaced with rat counterparts.

Ribosomal L10-L7/L12 protein complex and L11 bind to a highly conserved RNA region around position 1070 in domain II of 23 S rRNA and constitute a part of the GTPase-associated center in Escherichia coli ribosomes. We replaced these ribosomal proteins in vitro with the rat counterparts P0-P1/P2 complex and RL12, and tested them for ribosomal activities. The core 50 S subunit lacking the proteins on the 1070 RNA domain was prepared under gentle conditions from a mutant deficient in ribosomal protein L11. The rat proteins bound to the core 50 S subunit through their interactions with the 1070 RNA domain. The resultant hybrid ribosome was insensitive to thiostrepton and showed poly(U)-programmed polyphenylalanine synthesis dependent on the actions of both eukaryotic elongation factors 1alpha (eEF-1alpha) and 2 (eEF-2) but not of the prokaryotic equivalent factors EF-Tu and EF-G. The results from replacement of either the L10-L7/L12 complex or L11 with rat protein showed that the P0-P1/P2 complex, and not RL12, was responsible for the specificity of the eukaryotic ribosomes to eukaryotic elongation factors and for the accompanying GTPase activity. The presence of either E. coli L11 or rat RL12 considerably stimulated the polyphenylalanine synthesis by the hybrid ribosome, suggesting that L11/RL12 proteins play an important role in post-GTPase events of translation elongation.     

Cross-linking of elongation factor 2 to rat-liver ribosomal proteins by 2-iminothiolane

Complexes containing rat liver 80S ribosomes treated with puromycin and high concentrations of KCl, elongation factor 2 (EF-2) from pig liver, and guanosine 5'-[beta, gamma-methylene]triphosphate were prepared. Neighboring proteins in the complexes were cross-linked with the bifunctional reagent 2-iminothiolane. Proteins were extracted and then separated into 22 fractions by chromatography on carboxymethylcellulose of which seven fractions were used for further analyses. Each protein fraction was subjected to diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Nine cross-linked protein pairs between EF-2 and ribosomal proteins were shifted from the line formed with monomeric proteins. The spots of ribosomal proteins cross-linked to EF-2 were cut out from the gel plate and labelled with 125I. The labelled protein was extracted from the gel and identified by three kinds of two-dimensional gel electrophoresis, followed by autoradiography. The following proteins of both large and small subunits were identified: L9, L12, L23, LA33 (acidic protein of Mr 33000), P2, S6 and S23/S24, and L3 and L4 in lower yields. The results are discussed in relation to the topographies of ribosomal proteins in large and small subunits. Furthermore we found new neighboring protein pairs in large subunits, LA33-L11 and LA33-L12.     

Study of the surface of Escherichia coli ribosomes and ribosomal particles by the tritium bombardment method

A new technique of atomic tritium bombardment has been used to study the surface topography of Escherichia coli ribosomes and ribosomal subunits. The technique provides for the labeling of proteins exposed on the surface of ribosomal particles, the extent of protein labeling being proportional to the degree of exposure. The following proteins were considerably tritiated in the 70S ribosomes: S1, S4, S7, S9 and/or S11, S12 and/or L20, S13, S18, S20, S21, L1, L5, L6, L7/L12, L10, L11, L16, L17, L24, L26 and L27. A conclusion is drawn that these proteins are exposed on the ribosome surface to an essentially greater extent than the others. Dissociation of 70S ribosomes into the ribosomal subunits by decreasing Mg2+ concentration does not lead to the exposure of additional ribosomal proteins. This implies that there are no proteins on the contacting surfaces of the subunits. However, if a mixture of subunits has been subjected to centrifugation in a low Mg2+ concentration at high concentrations of a monovalent cation, proteins S3, S5, S7, S14, S18 and L16 are more exposed on the surface of the isolated 30S and 50S subunits than in the subunit mixture or in the 70S ribosomes. The exposure of additional proteins is explained by distortion of the native quaternary structure of ribosomal subunits as a result of the separation procedure. Reassociation of isolated subunits at high Mg2+ concentration results in shielding of proteins S3, S5, S7 and S18 and can be explained by reconstitution of the intact 30S subunit structure.     

Ribosomal Heterogeneity of Maize Tissues: Insights of Biological Relevance

In recent years, the selective role of ribosomes in the translational process of eukaryotes has been suggested. Evidence indicates that ribosomal heterogeneity at the level of protein stoichiometry and phosphorylation status differs among organisms, suggesting ribosomal specialization according to the state of development and the surrounding environment. During germination, protein synthesis is an active process that begins with the translation of the mRNAs stored in quiescent seeds and continues with the newly synthesized mRNAs. In this study, we identified differences in the abundance of ribosomal proteins (RPs) in maize embryos at different developmental stages. The relative quantification of RPs during germination revealed changes in six small subunit proteins, S3 (uS3), S5 (uS7), S7 (eS7), two isoforms of S17 (eS17), and S18 (uS13), and nine large subunit proteins, L1 (uL1), L5 (uL18), two isoforms of P0 (uL10), L11 (uL5), L14 (eL14), L15 (eL15), L19 (eL19), and L27 (eL27). Further analysis of ribosomal protein phosphorylation during germination revealed that the phosphorylation of PRP0 (uL10) and P1 increased and that of PRS3 (uS3) decreased in germinated versus quiescent embryos. The addition of insulin during germination increased the phosphorylation of the P1 protein, suggesting that its phosphorylation is controlled by the TOR pathway. Our results indicate that a heterogeneous ribosomal population provides to maize ribosomes during germination a different ability to translate mRNAs, suggesting another level of regulation by the ribosomes.     

Characterisation of the ribosomal binding site for eukaryotic elongation factor 2 by chemical cross-linking

Ribosomal complexes containing elongation factor 2 (EF-2) were formed by incubation of 80 S ribosomes in the presence of EF-2 and the non-hydrolysable GTP analogue GuoPP[CH2]P. The factor was covalently coupled to the ribosomal proteins located at the factor binding site, by treatment with bifunctional reagents. After isolation of the covalent EF-2.ribosomal protein complexes, the proteins were labelled with 125I and the introduced covalent links cleaved. The ribosomal proteins were identified by electrophoresis in two independent two-dimensional gel systems, followed by autoradiography. After cross-linking with bis(hydroxysuccinimidyl) tartrate (4 A between the reactive groups), protein S3/S3a, S7 and S11 were found as the major ribosomal proteins covalently linked to EF-2. The longer reagent, dimethyl 3,8-diaza-4,7-dioxo-5,6-dihydroxydecanbisimidate (11 A between the reactive groups), covalently coupled proteins S7, S11, L5, L13, L21, L23, L26, L27a and L32 to EF-2. After cross-linking with dimethyl suberimidate (9 A between the reactive groups) proteins S3/3a, S7, S11, L5, L8, L13, L21, L23, L26, L27a, L31 and L32 were identified as belonging to the EF-2-binding site. The results indicate that the ribosomal domain interacting with EF-2 is located on both the small and the large ribosomal subunit close to the subunit interface.     

Trypanosoma brucei mitochondrial ribosomes: affinity purification and component identification by mass spectrometry

Although eukaryotic mitochondrial (mt) ribosomes evolved from a putative prokaryotic ancestor their compositions vary considerably among organisms. We determined the protein composition of tandem affinity-purified Trypanosoma brucei mt ribosomes by mass spectrometry and identified 133 proteins of which 77 were associated with the large subunit and 56 were associated with the small subunit. Comparisons with bacterial and mammalian mt ribosomal proteins identified T. brucei mt homologs of L2-4, L7/12, L9, L11, L13-17, L20-24, L27-30, L33, L38, L43, L46, L47, L49, L52, S5, S6, S8, S9, S11, S15-18, S29, and S34, although the degree of conservation varied widely. Sequence characteristics of some of the component proteins indicated apparent functions in rRNA modification and processing, protein assembly, and mitochondrial metabolism implying possible additional roles for these proteins. Nevertheless most of the identified proteins have no homology outside Kinetoplastida implying very low conservation and/or a divergent function in kinetoplastid mitochondria.     

Study of the photoaffinity modification of Escherichia coli ribosomes near the donor tRNA-binding center

Affinity labelling of E. coli ribosomes near the donor tRNA-binding (P) site was studied with the use of photoreactive derivatives of tRNAPhe bearing arylazidogroups on N7 atoms of guanine residues (azido-tRNA). UV-irradiation of complexes 70S ribosome.poly(U).azido- tRNA(P-site) and 70S ribosome.poly(U).azido-tRNA(P-site).Phe- tRNAPhe(A-site) resulted in covalent attachment of azido-tRNA to ribosomes, both subunits being labelled. In both cases modification extent of 30S subunit was two-fold than that of the 50S one. It was shown that when the A-site was free the azido-tRNA located in P-site labelled proteins S9, S11, S12, S13, S21 and L14, L27, L31. Azido-tRNA located in P-site when the A-site was occupied with Phe-tRNAPhe labelled proteins S11, S12, S13, S14, S19, L32/L33 and possibly L23, L25. From the comparison of the sets of proteins labelled when A-site was free or occupied a conclusion was drawn that aminoacyl-tRNA located in ribosomal A-site affects the arrangement of deacylated tRNA in P-site. Data obtained allow to propose that proteins S5, S19, S20 and L24, L33 interact with guanine residues important for the tRNA tertiary structure formation.     

Structural arrangement of tRNA binding sites on Escherichia coli ribosomes, as revealed from data on affinity labelling with photoactivatable tRNA derivatives

A systematic study of protein environment of tRNA in ribosomes in model complexes representing different translation steps was carried out using the affinity labelling of the ribosomes with tRNA derivatives bearing aryl azide groups scattered statistically over tRNA guanine residues. Analysis of the proteins crosslinked to tRNA derivatives showed that the location of the derivatives in the aminoacyl (A) site led to the labelling of the proteins S5 and S7 in all complexes studied, whereas the labelling of the proteins S2, S8, S9, S11, S14, S16, S17, S18, S19, S21 as well as L9, L11, L14, L15, L21, L23, L24, L29 depended on the state of tRNA in A site. Similarly, the location of tRNA derivatives in the peptidyl (P) site resulted in the labelling of the proteins L27, S11, S13 and S19 in all states, whereas the labelling of the proteins S5, S7, S9, S12, S14, S20, S21 as well as L2, L13, L14, L17, L24, L27, L31, L32, L33 depended on the type of complex. The derivatives of tRNA(fMet) were found to crosslink to S1, S3, S5, S7, S9, S14 and L1, L2, L7/L12, L27. Based on the data obtained, a general principle of the dynamic functioning of ribosomes has been proposed: (i) the formation of each type of ribosomal complex is accompanied by changes in mutual arrangement of proteins - 'conformational adjustment' of the ribosome - and (ii) a ribosome can dynamically change its internal structure at each step of initiation and elongation; on the 70 S ribosome there are no rigidly fixed structures forming tRNA-binding sites (primarily A and P sites).     

Immunological studies of Escherichia coli mutants lacking one or two ribosomal proteins

Summary A battery of immunological tests were used to investigate mutants which had been determined as lacking one or two ribosomal proteins on the basis of two-dimensional polyacrylamide gels. Proteins which were confirmed as missing from the ribosome in one or more mutants were large subunit proteins L1, L15, L19, L24, L27, L28, L30 and L33 and small subunit proteins S1, S9, S17 and S20. Cross-reacting material (CRM) was also absent from the post-ribosomal supernatant except in the case of protein S1. Since mutants lacking protein L11 have been previously described, any one of 13 of the 52 ribosomal proteins can be missing. None of these 13 proteins, except S1, can therefore have an indispensable role in ribosome function or assembly. In several mutants in which a protein was not missing but altered, it was present as several moieties of differing charge and size.     

Determination of tRNA nucleotide residues directly interacting with proteins in the post- and pretranslocated ribosomal complexes

Nucleotide residues of E. coli tRNA interacting directly with proteins in pre- and posttranslocated ribosomal complexes have been identified by analysis of photo-induced tRNA-protein cross-links. A9, G18, A26 and U59 residues of NAcPhePhe-tRNA, located in the Ab-site (pretranslocated complex) have been cross-linked with proteins S10, L27, S7 and L2 respectively. In deacylated tRNA, located in the Pb-site, residues C17, G44, C56 and U60 have been cross-linked with proteins L2, L5, L27 and S9 respectively. The G44-L5 cross-link disappeared after translocation (NAc-PhePhe-tRNA located in the Pt-site).     

Are there proteins between the ribosomal subunits? Hot tritium bombardment experiments

The hot tritium bombardment technique [(1976) Dokl. Akad. Nauk SSSR 228, 1237-1238] was used for studying the surface localization of ribosomal proteins on Escherichia coli ribosomes. The degree of tritium labeling of proteins was considered as a measure of their exposure (surface localization). Proteins S1, S4, S7, S9 and/or S11, S12 and/or L20, S13, S18, S20, S21, L5, L6, L7/L12, L10, L11, L16, L17, L24, L26 and L27 were shown to be the most exposed on the ribosome surface. The sets of exposed ribosomal proteins on the surface of 70 S ribosomes, on the one hand, and the surfaces of 50 S and 30 S ribosomal subunits in the dissociated state, on the other, were compared. It was found that the dissociation of ribosomes into subunits did not result in exposure of additional ribosomal proteins. The conclusion was drawn that proteins are absent from the contacting surfaces of the ribosomal subunits.