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.     

Cross-linking study on protein topography of rat liver 60 S ribosomal subunits with 2-iminothiolane

Rat liver 60 S ribosomal subunits were modified with 2-iminothiolane. After treatment with hydrogen peroxide, the cross-linked proteins were extracted and then separated into 24 fractions by chromatography on carboxymethylcellulose. Each protein fraction was then analyzed by diagonal polyacrylamide-sodium dodecyl sulfate gel electrophoresis (Sommer, A., and Traut, R.R. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 3946-3950). The pieces of gel containing cross-linked protein spots that were shifted from the diagonal line were labeled with 125I. The labeled protein was extracted from the gel and identified by three kinds of two-dimensional gel electrophoresis, followed by autoradiography. Fifty-three cross-linked protein pairs involving 35 protein species containing two acidic proteins were identified. From these and previous results, a preliminary model of the protein topography of the 60 S ribosomal subunit was constructed and discussed in relation to other functional data on 60 S ribosomal proteins.     

Cross-linking study on protein neighborhoods at the subunit interface of rat liver ribosomes with 2-iminothiolane

Rat liver 80 S ribosomes were cross-linked with 2-iminothiolane. Proteins extracted from the cross-linked 80 S ribosomes were separated into 25 fractions by chromatography on carboxy methylcellulose. Each protein fraction was analyzed by diagonal polyacrylamide-sodium dodecyl sulfate gel electrophoresis. Eight pairs characteristic of 80 S ribosomes were detected which did not appear when isolated 40 S and 60 S subunits were cross-linked, and the cross-linked proteins were analyzed in similar manners. The cross-linked components were radioiodinated and then analyzed by two-dimensional gel electrophoresis, followed by autoradiography. Eight kinds of cross-links between 60 S subunit proteins and 40 S subunit proteins were identified as follows: SA30 (acidic protein with Mr 30,000)-LA33 (acidic protein with Mr 33,000), S2-LA33, S2-L11, S3a-L11, S4-L5, S25-L5, S4-L24 and S6-L24.     

Identification of the ribosomal proteins present in the vicinity of globin mRNA in the 40S initiation complex

The interaction of ribosomal proteins with mRNA in the 40S initiation complex was examined by chemical cross-linking. 40S initiation complexes were formed by incubating rat liver [(3)H]Met-tRNAi, rat liver 40S ribosomal subunits, rabbit globin mRNA, and partially purified initiation factors of rabbit reticulocytes in the presence of guanylyl(beta, gamma-methylene)-diphosphonate. The initiation complexes were then treated with 1,3-butadiene diepoxide to introduce crosslinks between the mRNA and proteins. The covalent mRNA-protein conjugates were isolated by chromatography on an oligo(dT) cellulose column in the presence of sodium dodecyl sulfate, followed by sucrose density gradient centrifugation. Proteins cross-linked to the mRNA were labeled with Na(125)I, extracted by extensive ribonuclease digestion, and analyzed by two-dimensional and diagonal polyacrylamide gel electrophoresis. Three ribosomal proteins, S6, S8, and S23/S24, together with small amounts of S3/S3a, S27, and S30, were identified as the protein components cross-linked to the globin mRNA protein complex, and were shown to attach directly to the mRNA. It is suggested that these proteins constitute the ribosomal binding site for mRNA in the 40S initiation complex.     

The subunit interface of the Escherichia coli ribosome: Identification of proteins at the interface between the 30 S and 50 S subunits by crosslinking with 2-iminothiolane

The 70 S ribosomes of Escherichia coli were treated with 2-iminothiolane with the resultant addition of 110 sulfhydryl groups per ribosome. The modified ribosomes were oxidized to promote disulfide bond formation, some of which formed intermolecular crosslinks. About 50% of the crosslinked 70 S ribosomes did not dissociate when exposed to low concentrations of magnesium in the absence of reducting agent. Dissociation took place in the presence of reducing agents, which indicated that the subunits had become covalently linked by disulfide linkages. Proteins extracted from purified crosslinked 70 S ribosomes were first fractionated by polyacrylamide/urea gel electrophoresis. The proteins from sequential slices of these gels were analyzed by two-dimensional polyacrylamide/sodium dodecyl sulfate diagonal gel electrophoresis. Monomeric proteins derived from crosslinked dimers appeared below the diagonal containing non-crosslinked proteins, since the second electrophoresis, but not the first, is run under reducing conditions to cleave the crosslinked species. Final identification of the proteins in each dimer was made by radioiodination of the crosslinked proteins, followed by two-dimensional polyacrylamide/urea gel electrophoresis in the presence of non-radioactive total 70 S proteins as markers. This paper describes the identification of 23 protein dimers that contained one protein from each of the two different ribosomal subunits. The proteins implicated must have some part of their structure in proximity to the other ribosomal subunit and are therefore defined as “interface proteins”. The group of interface proteins thus defined includes 50 S proteins that are part of the 5 S RNA: protein complex and 30 S proteins at the initiation site. Correlations between the crosslinked interface proteins and other functional data are discussed.     

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.     

Levels of ribosomal protein S1 and elongation factor G in the growth cycle of Escherichia coli.

The relative levels of ribosomes, ribosomal protein S1, and elongation factor G in the growth cycle of Escherichia coli were examined with two-dimensional polyacrylamide gel electrophoresis. Nonequilibrium pH gradient polyacrylamide gel electrophoresis was used in the first dimension, and polyacrylamide gradient-sodium dodecyl sulfate gel electrophoresis was used in the second dimension. The identities of protein spots containing S1 and elongation factor G were confirmed by radioiodination of the proteins and peptide mapping of the radiolabeled peptides. The levels of ribosomes and ribosomal protein S1 were coordinately reduced during transition from exponential phase to stationary phase. There was no accumulation of S1 in the stationary phase. In marked contrast, the level of elongation factor G showed no significant change from exponential phase to stationary phase. The relative level of elongation factor G compared with ribosomes or S1 increased by about 2.5-fold during transition from exponential phase to stationary phase. The results show that there are differences between the regulation of the levels of elongation factor G and of ribosomal proteins, including S1, apparent during the transition from exponential to stationary phase.     

Preparation of Escherichia coli 70S ribosomes labeled with 35S

[35S]--70S ribosomes (150 Ci/mmol) were isolated from E. coli MRE-600 cells grown on glucose-mineral media in the presence of [35S] ammonium sulfate. The labeled 30S and 50S subunits were obtained from [35S] ribosomes by centrifugation in a sucrose density gradient of 10--30% under dissociating conditions (0.5 mM Mg2+). The activity of [35S]--70S ribosomes obtained by reassociation of the labeled subunits during poly(U)-dependent diphenylalanine synthesis was not less than 70%. The activity of [35S]--70S ribosomes during poly(U)-directed polyphenylalanine synthesis was nearly the same as that of the standard preparation of unlabeled ribosomes. The 23S, 16S and 5S RNAs isolated from labeled ribosomes as total rRNA contained no detectable amounts of their fragments as revealed by polyacrylamide gel electrophoresis. The [35S] ribosomal proteins isolated from labeled ribosomes were analyzed by two-dimensional gel electrophoresis. The [35S] label was found in all proteins, with the exception of L20, L24 and L33 which did not contain methionine or cysteine residues.     

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.     

A comparative study on 40S ribosomal proteins of Artemia salina and rat liver: micro analysis of amino acid composition by high-performance liquid chromatography

The amino acid compositions of 24 proteins of 40S ribosomal subunits of Artemia salina cysts were determined and compared with those of rat liver. The basic proteins of A. salina 40S ribosomes were separated by two-dimensional polyacrylamide gel electrophoresis and extracted with 70% formic acid. Samples were freed from contaminants by gel-filtration through a high-performance liquid chromatography column. Amino acid compositions were determined for individual proteins by pre-column derivatization with N,N-dimethylaminoazobenzenesulfonyl chloride followed by reverse phase high-performance liquid chromatography. The similarity of amino acid compositions between A. salina and rat liver 40S ribosomal proteins was evaluated by the method of Cornish-Bowden (Cornish-Bowden, A. (1980) Anal. Biochem. 105, 233-238), and possible relationships between A. salina and rat were detected for 16 protein species (S2, S3, S4, S6, S7, S8, S15a, S16, S17, and S18, strongly related and S14, S15, S20, S23, S24, and S26, weakly related), indicating a conservative nature of eukaryotic ribosomal proteins.     

Protein topography of Sulfolobus solfataricus ribosomes by cross-linking with 2-iminothiolane. Sso L12e, Sso L10e, and Sso L11e are neighbors

Large ribosomal subunits from Sulfolobus solfataricus were cross-linked with 2-iminothiolane in order to investigate the arrangement of proteins in the region containing the multicopy acidic protein Sso L12e, the protein homologous to Escherichia coli L7/L12. Proteins from cross-linked 50 S subunits were extracted and fractionated by chromatography on CM-cellulose. Fractions containing Sso L12e were analyzed by "diagonal" (two-dimensional reducing/nonreducing) dodecyl sulfate polyacrylamide gel electrophoresis. Sso L12e appeared in cross-linked homodimers and also in cross-linked complexes that contained Sso L10e, the protein equivalent to E. coli L10. In addition, Sso L12e was found in cross-links to L4, L6a, L26, and L29. N-terminal sequences obtained for L6a and L26 showed them to have significant homologies to E. coli proteins L11 and L23, respectively. The results indicate the presence in this archaebacterial ribosome of Sso L12e dimers and their location near Sso L10e and Sso L11e. The Sso L12e-L29 (Sso L23e) cross-link suggests proximity between components of the factor-binding and peptidyltransferase domains, since E. coli L23 is a protein affinity-labeled by puromycin. The (Sso L12e)4-Sso L10 pentameric complex, identified previously from studies in solution, appears to represent correctly the arrangement of these proteins in the ribosome. The occurrence in the archaebacterial ribosome of this unique structural element, similar to those shown previously in eubacteria and eukaryotes, reinforces the concept that the protein quaternary structure of the ribosomal factor-binding domain is highly conserved.     

Antibiotic effects on the photoinduced affinity labeling of Escherichia coli ribosomes by puromycin.

The effect of ribosomal antibiotics on the photoinduced affinity labeling of Escherichia coli ribosomes by puromycin [Cooperman, B.S., Jaynes, E.N., Brunswick, D.J., & Luddy, M.A. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 1974; Jaynes, E.N. Jr., Grant, P.G., Giangrande, G., Wieder, R., & Cooperman, B.S. (1978) Biochemistry 17, 561] has been studied. Although blasticidin S, sparsomycin, lincomycin, and erythromycin are essentially without effect, major changes are seen on addition of either chloramphenicol or tetracycline. The products of photoincorporation have been characterized by one- and two-dimensional gel electrophoresis and by specific immunoprecipitation with antibodies to ribosomal proteins. In the presence of chloramphenicol, protein S14 becomes the major labeled protein. In the presence of tetracycline, L23 remains the major labeled protein, but the yield of labeled ribosomes is enormously increased, and the labeling is more specific for L23. These results are discussed in terms of the known modes of action of these antibiotics and the photoreactivity of tetracycline.     

Identification of the protein cross-linked to 3′-terminus of 5 S RNA in rat liver ribosomal 60 S subunits

To cross-link the 3′-terminus of 5 S RNA to its neighbouring proteins, ribosomal 60 S subunits of rat liver were oxidized with sodium periodate and reduced with sodium borohydride. 5 S RNP was then isolated by EDTA treatment followed by sucrose density-gradient centrifugation and subjected to SDS-polyacrylamide gel electrophoresis. The protein with a slower mobility than the L5 protein, which was thought to be cross-linked 5 S RNP, was labeled with ¹²⁵I, treated with RNAase, and analyzed by two-dimensional polyacrylamide gel electrophoresis, followed by radioautography. A radioactive spot located anodically from L5 protein was observed, suggesting that it is the L5 protein-oligonucleotide complex. When analyzed by SDS slab polyacrylamide gel electrophoresis followed by radioautography, the peptide pattern of the α-chymotrypsin digest of this ¹²⁵I-labeled protein-oligonucleotide complex was similar to that of the digest of ¹²⁵I-labeled L5 protein. The results indicate that L5 protein binds to the 3′-terminal region of 5 S RNA in rat liver 60 S subunits.     

An analysis of alterations in ribosomal conformation using reductive methylation

Optimal conditions for reductive alkylation of ribosomal proteins in their native and denatured states were examined. The relative accessibility of rat liver ribosomal proteins to reductive alkylation was then examined. Intact ribosomes were firs labeled with [14C]formaldehyde and NaBH4. The proteins were then separated from RNA, denatured in 6 M guanidine, and labeled again using formaldehyde and NaB3H4. The relative accessibility of individual proteins to labeling in the intact state could thus be determined from their 3H/14C ratios following separation by two-dimensional electrophoresis. The results suggest that proteins S6, S11, S26, L3, and L35 are less accessible to labeling while proteins S1, S15, L11, L12, L16, and L24 appear relatively more accessible. The accessibility of individual proteins in ribosomes in different conformational states were then compared. The results indicated that S3, L7, and L36 are likely to be involved in a structural difference when normal polysomes and normal monomers are compared. Also, that S26 and L35, and probably S3, S20, L7, L8, L24, L27, L28 and L34 appear to be involved in a ribosomal conformation change induced by ethionine intoxication.     

Identification by RNA-protein cross-linking of ribosomal proteins located at the interface between the small and the large subunits of mammalian ribosomes

Protein constituents at the subunit interface of rat liver ribosomes were analysed by cross-linking with the bifunctional reagent, diepoxybutane (distance between reactive groups 4 A). Isolated 40S and 60S subunits were labelled with 125I and recombined with unlabelled complementary subunits. The two kinds of selectively labelled 80S ribosomes were treated with diepoxybutane at low concentration. Radioactive ribosomal proteins covalently attached to the rRNA of the unlabelled complementary subparticles were isolated by repeated gradient centrifugation. The RNA-bound, labelled proteins were identified by two-dimensional gel electrophoresis. The experiments showed that proteins S2, S3, S4, S6, S7, S13, and S14 in the small subunit of rat liver ribosomes are located at the ribosomal interface in close proximity to 28S rRNA. Similarly, proteins L3, L6, L7, and L8 were found at the the interface of the large ribosomal subunit in the close vicinity of 18S rRNA.     

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.     

Group fractionation and determination of the number of ribosomal subunit proteins from Drosophila melanogaster embryos

Proteins were extracted from ribosomes and (for the first time) from ribosomal subunits of Drosophila melanogaster embryos. The ribosomal proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis. The electrophoretograms displayed 78 spots for the 80S monomers, 35 spots for the 60S subunits, and 31 spots for the 40S subunits. On the basis of present information, we propose what we believe to be a reliable and convenient nomenclature for the proteins of the ribosomes and each of the subunits. A pair of acidic proteins from D. melanogaster appears to be very similar in electrophoretic mobility to the acidic proteins L7/L12 from Escherichia coli and L40/L41 from rat liver. The electrophoretogram of proteins from embryonic ribosomes shows both qualitative and quantitative differences from those of larvae, pupae, and adults previously reported by others. The proteins of the 40S subunit range in molecular weight from approximately 10,000 to 50,000, and those from the 60S subunit range from approximately 11,000 to 50,000.     

Identification of neighboring protein pairs in the 60 S ribosomal subunits from Saccharomyces cerevisiae by chemical cross-linking

Protein-protein cross-linking was used to determine the spatial arrangement of proteins within the 60 S ribosomal subunits of Saccharomyces cerevisiae. Protein cross-links were generated by treatment of intact ribosomal subunits with dimethyl 3,3'-dithiobispropionimidate. Proteins were extracted from the treated subunits and fractionated by Cm-cellulose chromatography. Cross-linked proteins in these fractions were analyzed by electrophoresis on two-dimensional diagonal polyacrylamide gels containing sodium dodecyl sulfate. Component members of cross-linked pairs were radiolabeled with 125I and identified by two-dimensional gel electrophoresis and comparison with nonradioactive ribosomal protein markers. Seventeen pairs involving 16 of the 45 60 S subunit proteins were identified. Several proteins were detected in numerous cross-linked dimers and were used as foci for constructing a model depicting the arrangement of proteins within the 60 S ribosomal subunit. The model also incorporated previously published data on structure and function of proteins from the yeast 60 S subunit.     

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.     

Selective high-efficiency cross-linking of mammalian ribosomal proteins with cleavable thiol-directed heterobifunctional reagents: separation and identification of contact sequences of neighboring proteins after CNBr fragmentation

Mammalian ribosomal proteins were cross-linked in situ with the primarily cysteine-selective heterobifunctional reagents N-succinimidyl 2-(4-hydroxy-2-maleimidophenylazo)benzoate (reagent A, maximum range approx. 8 A) and N-succinimidyl 4-(4-hydroxy-3-maleimidophenylazo)[carboxyl-14C]benzoate (reagent B, maximum range approx. 12 A). With reagent B the secondarily attached (N-aryolated) protein becomes labelled specifically at the receptor amino group (lysine). The cross-linked proteins were fragmented with CNBr in attempts to isolate and identify sequences involved in the next-neighbor contacts. Two experimental schemes were adopted. Heavy complexes containing the large protein L4 cross-linked to protein L14 and/or L18 were isolated and treated with CNBr. The split products were submitted to diagonal electrophoresis for separation and identification of the two pairs of contact fragments. Proteins cross-linked with the radiolabelled reagent B were submitted to diagonal electrophoresis. The labelled receptor proteins were excised and treated with CNBr. Fragments carrying the contact sequences were separated by gradient gel electrophoresis and identified by autoradiography. By use of these methods CNBr fragments were isolated containing one or the dual contact sites of the following binary protein complexes: L4-L14, L4-L18, L4-L13a/L18a, L6'-L23, L6-L29, L7-L29, L14-L13a, L21-L18a, and L27-L30 (asterisks indicate the labelled receptor proteins). By varying the site of labelling of the heterobifunctional reagents and the methods of protein fragmentation a complete analysis of the contact sequences of these proteins should be possible.