Reconstitution of the active rat liver 60 S ribosomal subunit from different preparations of core particles and split proteins

Proteins extracted from the 60 S rat liver ribosomal subunit with 50% ethanol/0.5 M K Cl produced only a partial reactivation of the corresponding core particles. In contrast, the same split proteins were able to reactivate the core particles prepared with dimethyl-maleic anhydride (DMMA) to the same level as that observed using the DMMA-split proteins, i.e. 60-80% of the control according to the catalytic activities tested. Comparative analysis of the two split protein fractions showed only four common proteins: P1-P2, which alone restored part of the activities, especially the EF-2-dependent GTPase one, and L10a, L12, which must be responsible for the additional reactivation. The poor ability of the ethanol/KCl core particles to be reactivated was shown to be probably related to a conformational alteration which destabilized the 5 S RNA-protein complex. Proteins present in the ethanol/KCl wash of Saccharomyces cerevisiae 60 S subunits were found to be partly active in subunit reconstitution using rat liver DMMA core particles.     

Reconstitution and crystallisation experiments with isolated split proteins from Bacillus stearothermophilus ribosomes

Six proteins (B-L1, B-L6, B-L10, B-L11, B-L12 and B-L16) were removed from 50S ribosomal subunits of Bacillus stearothermophilus by treatment with ethanol and ammonium chloride. The proteins were isolated in a pure form, and one of them (B-L6) was crystallized. Five of the six proteins (in various combinations) were added back to the core particles, resulting in 50S subunits lacking one protein. The biological activities of these ribosomal particles as determined in the poly(U)-system varied over a wide range, depending on the protein which was omitted. The particles lacking one protein provide useful tools for heavy-atom derivation necessary for our crystallographic studies on the 50S subunits of Bacillus stearothermophilus.     

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.     

Puromycin binding to the small subunit of Escherichia coli ribosomes. Localization of the antibiotic in subunits reconstituted with puromycin-modified components

Small (30 S) ribosomal subunits from Escherichia coli strain TPR 201 were photoaffinity-labeled with [3H]puromycin in the presence of chloramphenicol under conditions in which more than 1 mol of antibiotic was incorporated per mol of ribosomes. The subunits were than washed with 3 M NH4Cl to yield core particles and a split protein fraction; the split proteins were further fractionated with ammonium sulfate. Subunits were then reconstituted using one fraction (core, split proteins, or ammonium sulfate supernatant) from photoaffinity-modified subunits and other components from unmodified (control) subunits. The distribution of [3H]puromycin in ribosomal proteins was monitored by one-dimensional polyacrylamide gel electrophoresis, and the sites of puromycin binding were visualized by immunoelectron microscopy. Two areas of puromycin binding were identified. A high affinity puromycin site, found on the upper third of the subunit and distant from the platform, is identical to the primary site previously identified (Olson, H. M., Grant, P. G., Glitz, D. G., and Cooperman, B. S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 890-894). Binding at this site is maximal in subunits reconstituted with high levels of puromycin-modified protein S14, and is decreased when unmodified S14 is incorporated. Because the percentage of antibody binding at the primary site always exceeds the percentage of puromycin label in protein S14, the primary site must include components other than S14. A secondary puromycin site of lower affinity is found on the subunit platform. This site is enriched in subunits reconstituted from puromycin-modified core particles and may include protein S7. Our results demonstrate the feasibility of localizing specifically modified components in reconstituted ribosomal subunits.     

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.     

Identification of proteins crosslinked to RNA in 40S ribosomal subunits of Saccharomyces cerevisiae

RNA-protein crosslinks were introduced into the 40S ribosomal subunits from Saccharomyces cerevisiae by mild UV treatment. Proteins crosslinked to the 18S rRNA molecule were separated from free proteins by repeated extraction of the treated subunits and centrifugation in glycerol gradients. After digestion with RNase to remove the RNA molecules, proteins were radio-labeled with 125I and identified by electrophoresis on two-dimensional polyacrylamide gels with carrier total 40S ribosomal proteins and autoradiography. Proteins S2, S7, S13, S14, S17/22/27, and S18 were linked to the 18S rRNA. A shorter period of irradiation resulted in crosslinking of S2 and S17/22/27 only. Several of these proteins were previously demonstrated to be present in ribosomal core particles or early assembled proteins.     

Identification of proteins crosslinked to RNA in 40S ribosomal subunits of Saccharomyces cerevisiae

RNA-protein crosslinks were introduced into the 40S ribosomal subunits from Saccharomyces cerevisiae by mild UV treatment. Proteins crosslinked to the 18S rRNA molecule were separated from free proteins by repeated extraction of the treated subunits and centrifugation in glycerol gradients. After digestion with RNase to remove the RNA molecules, proteins were radio-labeled with ¹²⁵I and identified by electrophoresis on two-dimensional polyacrylamide gels with carrier total 40S ribosomal proteins and autoradiography. Proteins S2, S7, S13, S14, S17/22/27, and S18 were linked to the 18S rRNA. A shorter period of irradiation resulted in crosslinking of S2 and S17/22/27 only. Several of these proteins were previously demonstrated to be present in ribosomal core particles or early assembled proteins.     

Partial reassembly of yeast 60 S ribosomal subunits in vitro following controlled dissociation under nondenaturing conditions

Previously it has been shown that 12 of the yeast ribosomal proteins were extractable from 60 S subunits under a specific nondenaturing condition [J. C. Lee, R. Anderson, Y. C. Yeh, and P. Horowitz (1985) Arch. Biochem. Biophys. 237, 292-299]. In the present paper, we showed that these proteins could be reassembled with the corresponding protein-deficient core particles to form biologically active ribosomal subunits. Effects of time, temperature, and varying concentrations of monovalent cations, divalent cations, cores, and ribosomal proteins on reconstitution were examined. Reconstitution was determined by binding of radiolabeled proteins to the nonradiolabeled cores as well as activity for polypeptide synthesis in a cell-free protein-synthesizing system. The optimal conditions for reconstitution were established. Whereas the core particles were about 10-20% as active as native 60 S subunits in an in vitro yeast cell-free protein-synthesizing system, the reconstituted particles were 80% as active. The activity of the reconstituted particles was proportional to the amount of extracted proteins added to the reconstitution mixture. About 55 +/- 7% of the core particles recombined with the extracted proteins to form reconstituted particles. These reconstituted particles cosedimented with native 60 S subunits in glycerol gradients and contained all of the 12 extractable proteins.     

Disassembly and reconstitution of yeast 60S ribosomal subunits

Summary Yeast 60S ribosomal subunits have been dissociated by reversible modification with dimethylmaleic anhydride. Treatment with 40 mol reagent/ml releases 35% of the protein, producing core particles inactive in polyphenylalanine synthesis, which are totally or highly deficient in 17 different proteins. This preparation of residual particles recovers 45% of the original activity upon incubation with the released proteins. The reconstituted particles can be isolated by centrifugation without loss of activity, having the protein composition of the original subunits.Abbreviations DMMA Dimethylmaleic Anhydride     

Identification of a ribosomal ATPase in Escherichia coli cells

Eukaryotic ribosomes harbor an ATPase activity that has been shown to be essential for translation elongation in some lower fungi. Here we report the first identification of a ribosome bound ATPase, RbbA, in E. coli cells. RbbA accounts for most of the ATPase activity associated with 70S ribosomes and 30S ribosomal subunits. Both native and recombinant RbbA were purified and shown to possess ribosome-dependent ATPase activities and to stimulate polyphenylalanine synthesis in vitro. Biochemically, RbbA is similar to the fungi-specific translation elongation factor 3 (EF-3) and cross-reacts with antibody raised against EF-3. The gene encoding RbbA is identified as ORF yhih and the predicted RbbA amino acid sequence is 40% similar to that of the C-terminal half of EF-3. The discovery of a ribosomal ATPase in a prokaryotic cell suggests a common, conserved function for these proteins in translation.     

Characterization of the ribosomal properties required for formation of a GTPase active complex with the eukaryotic elongation factor 2

The binding stability of the different nucleotide-dependent and -independent interactions between elongation factor 2 (EF-2) and 80S ribosomes, as well as 60S subunits, was studied and correlated to the kinetics of the EF-2- and ribosome-dependent hydrolysis of GTP. Empty reconstituted 80S ribosomes were found to contain two subpopulations of ribosomes, with approximately 80% capable of binding EF-2.GuoPP[CH2]P with high affinity (Kd less than 10(-9) M) and the rest only capable of binding the factor-nucleotide complex with low affinity (Kd = 3.7 x 10(-7) M). The activity of the EF-2- and 80S-ribosome dependent GTPase did not respond linearly to increasing factor concentrations. At low EF-2/ribosome ratios the number of GTP molecules hydrolyzed/factor molecule was considerably lower than at higher ratios. The low response coincided with the formation of the high-affinity complex. At increasing EF-2/ribosome ratios, the ribosomes capable of forming the high-affinity complex was saturated with EF-2, thus allowing formation of the low-affinity ribosome.EF-2 complex. Simultaneously, the GTPase activity/factor molecule increased, indicating that the low-affinity complex was responsible for activating the GTP hydrolysis. The large ribosomal subunits constituted a homogeneous population that interacted with EF-2 in a low-affinity (Kd = 1.3 x 10(-6) M) GTPase active complex, suggesting that the ribosomal domain responsible for activating the GTPase was located on the 60S subunit. Ricin treatment converted the 80S particles to the type of conformation only capable of interacting with EF-2 in a low-affinity complex. The structural alteration was accompanied by a dramatic increase in the EF-2-dependent GTPase activity. Surprisingly, ricin had no effect on the factor-catalyzed GTP hydrolysis in the presence of 60S subunits alone.     

Phosphorylation of wheat germ initiation factors and ribosomal proteins

The ability of the wheat germ initiation factors and ribosomes to serve as substrates for a wheat germ protein kinase (Yan and Tao 1982 J Biol Chem 257: 7037-7043) has been investigated. The wheat germ kinase catalyzes the phosphorylation of the 42,000 dalton subunit of eukaryotic initiation factor (eIF)-2 and the 107,000 dalton subunit of eIF-3. Other initiation factors, eIF-4B and eIF-4A, and elongation factors, EF-1 and EF-2, are not phosphorylated by the kinase. Quantitative analysis indicates that the kinase catalyzes the incorporation of about 0.5 to 0.6 mole of phosphate per mole of the 42,000 dalton subunit of eIF-2 and about 6 moles of phosphate per mole of the 107,000 dalton subunit of eIF-3. Three proteins (M_r = 38,000, 14,800, and 12,600) of the 60S ribosomal subunit are phosphorylated by the kinase, but none of the 40S ribosomal proteins are substrates of the kinase. No effects of phosphorylation on the activities of eIF-2, eIF-3, or 60S ribosomal subunits could be demonstrated in vitro.     

Mitochondrial ribosome assembly in Neurospora. Structural analysis of mature and partially assembled ribosomal subunits by equilibrium centrifugation in CsCl gradients

In Neurospora, one protein associated with the mitochondrial small ribosomal subunit (S-5, Mr 52,000) is synthesized intramitochondrially and is assumed to be encoded by mtDNA. When mitochondrial protein synthesis is inhibited, either by chloramphenicol or by mutation, cells accumulate incomplete mitochondrial small subunits (CAP-30S and INC-30S particles) that are deficient in S-5 and several other proteins. To gain additional insight into the role of S-5 in mitochondrial ribosome assembly, the structures of Neurospora mitochondrial ribosomal subunits, CAP-30S particles, and INC-30S particles were analyzed by equilibrium centrifugation in CsCl gradients containing different concentrations of Mg+2. The results show (a) that S-5 is tightly associated with small ribosomal subunits, as judged by the fact that it is among the last proteins to be dissociated in CsCl gradients as the Mg+2 concentration is decreased, and (b) that CAP-30S and INC-30S particles, which are deficient in S-5, contain at most 12 proteins that are bound as tightly as in mature small subunits. The CAP-30S particles isolated from sucrose gradients contain a number of proteins that appear to be loosely bound, as judged by dissociation of these proteins in CsCl gradients under conditions in which they remain associated with mature small subunits. The results suggest that S-5 is required for the stable binding of a subset of small subunit ribosomal proteins.     

Partial reconstitution of 60S ribosomal subunits from yeast

Summary Ribosomal 60S subunits active in polyphenylalanine synthesis can be reconstituted from core particles lacking 20–40% of the total protein. These core particles were obtained by treatment of yeast 60S subunits with dimethylmaleic anhydride, a reagent for protein amino groups. Upon reconstitution a complementary amount of split proteins is incorporated into the ribosomal particles, which have the sedimentation coefficient of the original subunits. Ribosomal protein fractions obtained by extraction with 1.25 M NH₄Cl, 4 M LiCl, 7 M LiCl, or 67% acetic acid, are much less efficient in the reconstitution of active subunits from these core particles than the corresponding released fraction prepared with dimethylmaleic anhydride. Attempts to reconstitute active subunits from protein-deficient particles obtained with 1.25 M NH₄Cl plus different preparations of ribosomal proteins, including the fraction released with dimethylmaleic anhydride, were unsuccessful. Therefore, under our conditions, of the disassembly procedures assayed only dimethylmaleic anhydride allows partial reconstitution of active 60S subunits.Abbreviation DMMA dimethylmaleic anhydride     

The binding site of ribosomal protein L10 in eubacteria and archaebacteria is conserved: reconstitution of chimeric 50S subunits.

It has been shown by electron microscopy that the selective removal of the stalk from 50S ribosomal subunits of two representative archaebacteria, namely Methanococcus vaniellii and Sulfolobus solfataricus, is accompanied by loss of the archaebacterial L10 and L12 proteins. The stalk was reformed if archaebacterial core particles were reconstituted with their corresponding split proteins. Next, structurally intact chimeric 50S subunits have been reconstituted in vitro by addition of Escherichia coli ribosomal proteins L10 and L7/L12 to 50S core particles from M vaniellii or S solfataricus, respectively. In the reverse experiment, using core particles from E coli and split proteins from M vaniellii, stalk-bearing 50S particles were also obtained. Analysis of the reconstituted 50S subunits by immunoblotting revealed that E coli L10 was incorporated into archaebacterial core particles in both presence or absence of E coli L7/L12. In contrast, incorporation of E coli L7/L12 into archaebacterial cores was only possible in the presence of E coli L10. Our results suggest that in archaebacteria - as in E coli - the stalk is formed by archaebacterial L12 proteins that bind to the ribosome via L10. The structural equivalence of eubacterial and archaebacterial L10 and L12 proteins has thus for the first time been established. The chimeric reconstitution experiments provide evidence that the domain of protein L10 that interacts with the ribosomal particle is highly conserved between eubacteria and archaebacteria.     

Translation elongation factor-3 (EF-3): An evolving eukaryotic ribosomal protein?

Fungi appear to be unique in their requirement for a third soluble translation elongation factor. This factor, designated elongation factor 3 (EF-3), exhibits ribosome-dependent ATPase and GTPase activities that are not intrinsic to the fungal ribosome but are nevertheless essential for translation elongation in vivo. The EF-3 polypeptide has been identified in a wide range of fungal species and the gene encoding EF-3 (YEF3) has been isolated from four fungal species (Saccharomyces cerevisiae, Candida albicans, Candida guillermondii, andPneumocystis carinii). Computer-assisted analysis of the predictedS. cerevisiae EF-3 amino acid sequence was used to identify several potential functional domains; two ATP binding/catalytic domains conserved with equivalent domains in members of the ATP-Binding Cassette (ABC) family of proteins, an aminoterminal region showing significant similarity to theE. coli S5 ribosomal protein, and regions of predicted interaction with rRNA, tRNA, and mRNA. Furthermore, EF-3 was also found to display amino acid similarity to myosin proteins whose cellular function is to provide the motive force of muscle. The identification of these regions provides clues to both the evolution and function of EF-3. The predicted functional regions are conserved among all known fungal EF-3 proteins and a recently described homologue encoded by the Chlorella virus CVK2. We propose that EF-3 may play a role in the ribosomal optimization of the accuracy of fungal protein synthesis by altering the conformation and activity of a ribosomal accuracy center, which is equivalent to the S4-S5-S12 ribosomal protein accuracy center domain of theE. coli ribosome. Furthermore, we suggest that EF-3 represents an evolving ribosomal protein with properties analogous to the intrinsic ATPase activities of higher eukaryotic ribosomes, which has wider implications for the evolutionary divergence of fungi from other eukaryotes. Correspondence to: M.F. Tuite     

Site of synthesis of spinach chloroplast ribosomal proteins and formation of incomplete ribosomal particles in isolated chloroplasts

Chloroplast ribosomal proteins from spinach have been prepared in the presence of a protease inhibitor and some modifications have been introduced to the previous characterization of the 50S subunits (Mache et al., MGG, 177, 333, 1980): 33 ribosomal proteins are detected instead of 34. No change has been observed for the 30S subunits.Using a light-driven system of protein synthesis it is shown that up to ten ribosomal proteins of the 30S and eight proteins of the 50S subunits are made in the chloroplast.Newly synthesized ribosomal subunits have been analysed on CsCl gradients after sedimentation at equilibrium, allowing the separation of fully assembled subunits from incomplete ribosomal particles. Most of the newly made 50S subunits are fully assembled (=1.634). A small amount of incomplete 50S particles (=1.686) is detectable. Newly made 30S subunits (=1.598) and incomplete 30S particles (=1.691) are also observed. The ribosomal proteins of the incomplete 30S have been determined. They contain eight or nine of the 30S-proteins, seven of which are synthesized within the chloroplast. It is suggested that incomplete ribosomal particles resulted from a step in the assembly of ribosomal subunits.     

Isolation and characterization of an inhibitor of ribosome-dependent GTP hydrolysis by elongation factor G

Two inhibitors of ribosome-dependent GTP hydrolysis by elongation factor (EF)G were found in the ribosome wash of Escherichia coli strain B. One of these inhibitors was purified to homogeneity and characterized. The isolated inhibitor was found to consist of two polypeptide subunits with apparent molecular masses of 23 kDa and 10 kDa. Inhibition of EF-G GTPase could not be overcome by increasing amounts of the elongation factor or high concentrations of GTP, but was reversed by large amounts of ribosomes. The effect of the inhibitor was reduced by increasing concentrations of either 30S or 50S ribosomal subunits. EF-G-dependent GTPase of 50S ribosomal subunits was not affected by the inhibitor. These findings clearly show that the inhibitor interferes with the modulation of EF-G GTPase activity by the interactions between 30S and 50S ribosomal subunits. Under conditions, where 30S CsCl core particles are able to associate with 50S subunits and to stimulate EF-G GTPase, the effect of the inhibitor was considerably reduced when intact 30S ribosomal subunits were substituted by 30S CsCl core particles. This finding indicates that 30S CsCl split proteins are important for the action of the inhibitor and that the inhibitor does not affect the EF-G GTPase merely by interfering with the association of ribosomal subunits. Furthermore, poly(U)-dependent poly(phenylalanine) synthesis was considerably less sensitive to the inhibitor than EF-G GTPase. When ribosomes were preincubated with poly(U) and Phe-tRNA(Phe), poly(phenylalanine) synthesis was considerably less affected by the inhibitor, whereas EF-G GTPase was still sensitive.     

Comparison of phosphorylation of ribosomal proteins from HeLa and Krebs II ascites-tumour cells by cyclic AMP-dependent and cyclic GMP-dependent protein kinases.

Phosphorylation of eukaryotic ribosomal proteins in vitro by essentially homogeneous preparations of cyclic AMP-dependent protein kinase catalytic subunit and cyclic GMP-dependent protein kinase was compared. Each protein kinase was added at a concentration of 30nM. Ribosomal proteins were identified by two-dimensional gel electrophoresis. Almost identical results were obtained when ribosomal subunits from HeLa or ascites-tumour cells were used. About 50-60% of the total radioactive phosphate incorporated into small-subunit ribosomal proteins by either kinase was associated with protein S6. In 90 min between 0.7 and 1.0 mol of phosphate/mol of protein S6 was incorporated by the catalytic subunit of cyclic AMP-dependent protein kinase. Of the other proteins, S3 and S7 from the small subunit and proteins L6, L18, L19 and L35 from the large subunit were predominantly phosphorylated by the cyclic AMP-dependent enzyme. Between 0.1 and 0.2 mol of phosphate was incorporated/mol of these phosphorylated proteins. With the exception of protein S7, the same proteins were also major substrates for the cyclic GMP-dependent protein kinase. Time courses of the phosphorylation of individual proteins from the small and large ribosomal subunits in the presence of either protein kinase suggested four types of phosphorylation reactions: (1) proteins S2, S10 and L5 were preferably phosphorylated by the cyclic GMP-dependent protein kinase; (2) proteins S3 and L6 were phosphorylated at very similar rates by either kinase; (3) proteins S7 and L29 were almost exclusively phosphorylated by the cyclic AMP-dependent protein kinase; (4) protein S6 and most of the other proteins were phosphorylated about two or three times faster by the cyclic AMP-dependent than by the cyclic GMP-dependent enzyme.     

Modification of the accessibility of ribosomal proteins after elongation factor 2 binding to rat liver ribosomes and during translocation

Free- and EF-2-bound 80 S ribosomes, within the high-affinity complex with the non-hydrolysable GTP analog: guanylylmethylenediphosphonate (GuoPP(CH2)P), and the low-affinity complex with GDP, were treated with trypsin under conditions that modified neither their protein synthesis ability nor their sedimentation constant nor the bound EF-2 itself. Proteins extracted from trypsin-digested ribosomes were unambiguously identified using three different two-dimensional gel electrophoresis systems and 5 S RNA release was checked by submitting directly free- and EF-2-bound 80 S ribosomes, incubated with trypsin, to two-dimensional gel electrophoresis. Our results indicate that the binding of (EF-2)-GuoPP[CH2]P to 80 S ribosomes modified the behavior of a cluster of five proteins which were trypsin-resistant within free 80 S ribosomes and trypsin-sensitive within the high-affinity complex (proteins: L3, L10, L13a, L26, L27a). As for the binding of (EF-2)-GDP to 80 S ribosomes, it induced an intermediate conformational change of ribosomes, unshielding only protein L13a and L27a. Quantitative release of free intact 5 S RNA which occurred in the first case but not in the second one, should be related to the trypsinolysis of protein(s) L3 and/or L10 and/or L26. Results were discussed in relation to structural and functional data available on the ribosomal proteins we found to be modified by EF-2 binding.