Detection of a membrane-associated protein on detached membrane ribosomes in Staphylococcus aureus

Membrane ribosomes from Staphylococcus aureus which were detached from the membrane by extraction with the nonionic detergent Triton X-100 retained a protein (MBRP) with a molecular weight of 60 000, which was absent from cytoplasmic ribosomes. MBRP was detected and quantified by immunological methods. When membrane ribosomes were dissociated into 50S and 30S subunits, MBRP remained associated with the 50S particle. MBRP was found both on membrane ribosomes and in the cytoplasm in roughly equal amounts. When added to Triton X-100-solubilized protoplasts, antibodies to MBRP produced immunoprecipitates which contained a complex of MBRP and three other proteins with molecular weights of 71 000, 46 000 and 41 000. Four proteins with the same molecular weights as those of the MBRP complex were found associated with membrane ribosomes. The proteins of molecular weight 71 000, 60 000, 46 000 and 41 000 seemed to be present in stoichiometrically equivalent amounts in the complex.     

Interaction of the cytoplasmic membrane and ribosomes in Escherichia coli; altered ribosomal proteins in sucrose-dependent spectinomycin-resistant mutants.

Alterations in the ribosomes of sucrose-dependent spectinomycin-resistant (Sucd-Spcr) mutants of Escherichia coli were studied. Subunit exchange experiments showed that 30S subunits were responsible for the resistance of ribosomes to spectinomycin in all Sucd-Spcr mutants tested. Proteins of 30S ribosomes were analyzed by carboxymethyl cellulose column chromatography based on their elution positions. Mutants YM22 and YM93 had an altered 30S ribosomal protein component, S5, and mutant YM50 had an altered protein, S4. Although a shift of elution position was not detected for all the 30S ribosomal proteins from mutant YM101, the amount of protein S3 was appreciably lowered in the isolated 30S subunits. A partial reconstitution experiment with protein S3 prepared from both the wild-type strain and YM101 revealed that the mutant had altered protein S3 which is responsible for the spectinomycin resistance. These alterations in 30S subunits are discussed in relation to the interaction between ribosomes and the cytoplasmic membrane.     

Identification of proteins involved in the peptidyl transferase activity of ribosomes by chemical modification.

Photochemical oxidation of Escherichia coli 50 S ribosomal subunits in the presence of methylene blue or Rose Bengal causes rapid loss of peptidyl transferase activity. Reconstitution experiments using mixtures of components from modified and unmodified ribosomes reveal that both RNA and proteins are affected, and that among the proteins responsible for inactivation there are both LiCl-split and core proteins. The proteins L2 and L16 from the split fraction and L4 from the core fraction of unmodified ribosomes were together nearly as effective as total unmodified proteins in restoring peptidyl transferase activity to reconstituted ribosomes when added with proteins from modified ribosomes. These three proteins are therefore the most important targets identified as responsible for loss of peptidyl transferase activity on photo-oxidation of 50 S ribosomal subunits.     

Immunoprecipitation of 70S, 50S and 30S ribosomes ofEscherichia coli

Antibodies were raised in rabbits against 70S ribosomes, 50S and 30S ribosomal subunits individually. Purified immunoglobulins from the antiserum against each of the above ribosomal entities were tested for their capabilities of precipitating 70S, 50S and 30S ribosomes. The observations revealed the following: (i) The antiserum (IgG) raised against 70S ribosomes precipitates 70S ribosomes completely, while partial precipitation is seen with the subunits, the extent of precipitation being more with the 50S subunits than with 30S subunits; addition of 50S subunits to the 30S subunits facilitates the precipitation of 30S subunits by the antibody against 70S ribosomes. (ii) Antiserum against 50S subunits has the ability to immunoprecipitate both 50S and 70S ribosomes to an equal extent. (iii) Antiserum against 30S subunits also has the property of precipitating both 30S and 70S ribosomes. The differences in the structural organisation of the two subunits may account for the differences in their immunoprecipitability.     

Surface topography of the Bacillus stearothermophilus ribosome.

Summary The surface topography of the intact 70S ribosome and free 30S and 50S subunits from Bacillus stearothermophilus strain 2184 was investigated by lactoperoxidase-catalyzed iodination. Two-dimensional polyacrylamide gel electrophoresis was employed to separate ribosomal proteins for analysis of their reactivity. Free 50S subunits incorporated about 18% more ¹²⁵I than did 50S subunits derived from 70S ribosomes, whereas free 30S subunits and 30S subunits derived from 70S ribosomes incorporated similar amounts of ¹²⁵I. Iodinated 70S ribosomes and subunits retained 62–78% of the protein synthesis activity of untreated particles and sedimentation profiles showed no gross conformational changes due to iodination. The proteins most reactive to enzymatic iodination were S4, S7, S10 and Sa of the small subunit and L2, L4, L5/9, L6 and L36 of the large subunit. Proteins S2, S3, S7, S13, Sa, L5/9, L10, L11 and L24/25 were labeled substantially more in the free subunits than in the 70S ribosome. Other proteins, including S5, S9, S12, S15/16, S18 and L36 were more extensively iodinated in the 70S ribosome than in the free subunits. The locations of tyrosine residues in some homologus ribosomal proteins from B. stearothermophilus and E. coli are compared.     

Changes in ribosome function induced by protein kinase associated with ribosomes of Streptomyces collinus producing kirromycin.

Protein kinase associated with ribosomes of streptomycetes phosphorylates 11 ribosomal proteins. Phosphorylation activity of protein kinase reaches its maximum at the end of exponential phase of growth. When (32)P-labeled cells from the end of exponential phase of growth were transferred to a fresh medium, after 2 h of cultivation ribosomal proteins lost more than 90% of (32)P and rate of polypeptide synthesis increases twice. Protein kinase cross-reacting with antibody raised against protein kinase C was partially purified from 1 M NH(4)Cl wash of ribosomes and used to phosphorylation of ribosomes. Phosphorylation of 50S subunits (L2, L3, L7, L16, L21, L23, and L27) had no effect on the integrity of subunits but affects association with 30 to 70S monosomes. In vitro system derived from ribosomal subunits was used to examine the activity of phosphorylated 50S at poly(U) translation. Replacement unphosphorylated 50S with 50S possessed of phosphorylated r-proteins leads to the reduction of polypeptide synthesis of about 52%. The binding of N-Ac[(14)C]Phe-tRNA to A-site of phosphorylated ribosomes is not affected but the rate of peptidyl transferase is more than twice lower than that in unphosphorylated ribosomes. These results provide evidence that phosphorylation of ribosomal proteins is involved in mechanisms regulating the translational system of Streptomyces collinus.     

Ribonuclease sensitivity of Escherichia coli ribosomes

Santer, Melvin (Haverford College, Haverford, Pa.), and Josephine R. Smith. Ribonuclease sensitivity of Escherichia coli ribosomes. J. Bacteriol. 92:1099-1110. 1966.-The ribonucleic acid (RNA) contained in 70S ribosomes and in 50S and 30S subunits was hydrolyzed by pancreatic ribonuclease. A 7% amount of the RNA was removed from the 70S particle; at 10(-4)m magnesium concentration, a maximum of 24 and 30% of the RNA in the 50S and the 30S fractions, respectively, was removed by ribonuclease. At the two lower magnesium ion concentrations, 50S ribosomes did not lose any protein, whereas 30S ribosomes lost protein as a result of ribonuclease treatment. A number of proteins were removed from the 30S particles by ribonuclease, and these proteins were antigenically related to proteins present in 50S ribosomes. The differential effect of ribonuclease on 50S and 30S ribosomes suggested that they have structural dissimilarities.     

Translation of Synthetic and Endogenous Messenger Ribonucleic Acid In Vitro by Ribosomes and Polyribosomes from Clostridium pasteurianum

Ribosomes and polyribosomes from Clostridium pasteurianum were isolated and their activities were compared with those of ribosomes from Escherichia coli in protein synthesis in vitro. C. pasteurianum ribosomes exhibited a high level of activity due to endogenous messenger ribonucleic acid (RNA). For translation of polyuridylic acid [poly(U)], C. pasteurianum ribosomes required a higher concentration of Mg(2+) and a much higher level of poly(U) than did E. coli ribosomes. Phage f2 RNA added to the system with C. pasteurianum ribosomes gave no significant stimulation of protein synthesis in a homologous system or with E. coli initiation factors. The 30S and 50S subunits prepared from C. pasteurianum ribosomes reassociated less readily than subunits from E. coli. The ability of the C. pasteurianum subunits to reassociated was found to be dependent upon the presence of a reducing agent during preparation and during analysis of the reassociation products. In heterologous combinations, E. coli 30S subunits associated readily with C. pasteurianum 50S subunits to form 70S particles, but C. pasteurianum 30S subunits and E. coli 50S subunits did not associate. In poly(U) translation, E. coli 30S subunits were active in combination with 50S subunits from either E. coli or C. pasteurianum, but C. pasteurianum 30S subunits were not active in combination with either type of 50S subunits. Polyribosomes prepared from C. pasteurianum were very active in protein synthesis, and well-defined ribosomal aggregates as large as heptamers could be seen on sucrose gradients. An attempt was made to demonstrate synthesis in vitro of ferredoxin.     

Reconstitution of active 50 S ribosomal subunits from Bacillus licheniformis and Bacillus subtilis.

Active 50 S ribosomal subunits from Bacillus licheniformis and Bacillus subtilis can be reconstituted in vitro from dissociated RNA and proteins. The reconstituted 50 S sub-units are indistinguishable from native 50 S subunits in sedimentation on sucrose gradients and in protein composition. The procedure used is similar to that developed for reconstitution of Bacillus stearothermophilus 50 S subunits, though the optimal conditions are somewhat different. Hybrid ribosomes can be reconstituted with 23 S RNA and proteins from different sources (B. stearothermophilus and B. licheniformis or B. subtilis). The thermal stability of these ribosomes depends on the source of the proteins, and not on the source of 23 S RNA.     

Determination of peptide regions on the surface of the eubacterial and archaebacterial ribosome by limited proteolytic digestion.

Limited proteolysis was used in combination with two-dimensional gel electrophoresis, blotting, and amino acid sequence analysis to investigate the surface of intact ribosomal subunits at the peptide and amino acid level. Surface sites of 14 ribosomal proteins from Escherichia coli 50S subunits were determined using proteases with different specificities. To assess the evolutionary conservation of ribosomal topography among eubacteria, large subunits from Bacillus stearothermophilus were also subjected to limited proteolysis. The results obtained indicate a conservation of the three-dimensional ribosomal structure at the peptide level. The data for the eubacterial ribosomes are in full agreement with the model of the 50S protein topography derived from immunological data. Furthermore, peptide surface regions of archaebacterial ribosomes have been investigated. The results presented in this work prove that limited proteolysis can successfully be applied to halophilic and thermophilic ribosomes from archaebacteria.     

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.     

Characterization of Ribosomes from Dormant Spores of Bacillus cereus

Characterization of ribosomes from dormant spores and vegetative cells of Bacillus cereus strain T has been carried out. Polyuridylic acid binding activity, ribonuclease activity associated with ribosomes, thermal denaturation profile, and sedimentation coefficients are essentially identical for both ribosomal preparations. However, ribosomal protein content of dormant spore ribosomes is about 70% of that of vegetative ribosomes. Polyacrylamide gel electrophoresis of ribosomal proteins shows that some ribosomal proteins are missing from dormant spore ribosomes. Sucrose density gradient centrifugation of ribosomes shows the existence of defective ribosomal subunits, in addition to 30S and 50S subunits, in dormant spore ribosomes. These results indicate that the ribosomes from dormant spores are distinctively different from those of vegetative cells.     

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.     

Membrane-bound ribosomes of myeloma cells. III. The role of the messenger RNA and the nascent polypeptide chain in the binding of ribosomes to membranes

Mild ribonuclease treatment of the membrane fraction of P3K cells released three types of membrane-bound ribosomal particles: (a) all the newly made native 40S subunits detected after 2 h of [3H]uridine pulse. Since after a 3-min pulse with [35S]methionine these membrane native subunits appear to contain at least sevenfold more Met-tRNA per particle than the free native subunits, they may all be initiation complexes with mRNA molecules which have just become associated with the membranes; (b) about 50% of the ribosomes present in polyribosomes. Evidence is presented that the released ribosomes carry nascent chains about two and a half to three times shorter than those present on the ribosomes remaining bound to the membranes. It is proposed that in the membrane-bound polyribosomes of P3K cells, only the ribosomes closer to the 3' end of the mRNA molecules are directly bound, while the latest ribosomes to enter the polyribosomal structures are indirectly bound through the mRNA molecules; (c) a small number of 40S subunits of polyribosomal origin, presumably initiation complexes attached at the 5' end of mRNA molecules of polyribosomes. When the P3K cells were incubated with inhibitors acting at different steps of protein synthesis, it was found that puromycin and pactamycin decreased by about 40% the proportion of ribosomes in the membrane fraction, while cycloheximide and anisomycin had no such effect. The ribosomes remaining on the membrane fraction of puromycin-treated cells consisted of a few polyribosomes, and of an accumulation of 80S and 60S particles, which were almost entirely released by high salt treatment of the membranes. The membrane-bound ribosomes found after pactamycin treatment consisted of a few polyribosomes, with a striking accumulation of native 60S subunits and an increased number of native 40S subunits. On the basis of the observations made in this and the preceding papers, a model for the binding of ribosomes to membranes and for the ribosomal cycle on the membranes is proposed. It is suggested that ribosomal subunits exchange between free and membrane-bound polyribosomes through the cytoplasmic pool of free native subunits, and that their entry into membrane-bound ribosomes is mediated by mRNA molecules associated with membranes.     

Two-dimensional gel electrophoresis of ribosomal proteins from streptomycin-sensitive and streptomycin-resistant mutants of Chlamydomonas reinhardi.

Ribosomal proteins from three mutant strains of Chlamydomonas reinhardi were analysed and compared by one-dimensional and two-dimensional gel electrophoresis. One mutant was streptomycin-sensitive the other two were streptomycin-resistant, one with a Mendelian the other with a non-Mendelian pattern of inheritance. In the 30-S subunits of chloroplast ribosomes approximately 25 proteins are found and in the 50-S subunits 34 proteins. The 40-S subunits of cytoplasmic ribosomes contain about 31 proteins and the 60-S subunits 44 proteins. The molecular weights of most proteins in all subunits are in the range of 10 000 to 35 000. However, the 60-S subunits contain in addition a protein of molecular weight 50 000 and the 30-S subunits show 6-7 bands of molecular weights from 50 000 to 83 000. The proteins of the cytoplasmic 80-S ribosomes or of their subunits from all three mutants are electrophoretically identical. The proteins of the 70-S organellar ribosomes and both of their subunits show distinct differences between the three strains. Our results indicate that organellar ribosomal proteins are in part controlled by nuclear DNA and in part by organellar DNA.     

Characterization of ribosomes of a strain ofStreptomyces aureofaciens producing chlortetracycline

These studies were designated to investigate the effect of chlortetracycline on sedimentation properties of polysomes and ribosomes present in the chlortetracycline producing strain ofStreptomyces aureofaciens. In presence of chlortetracycline polysomes and ribosomes are more stable than the bacterial ones. At lower chlortetracycline concentrations (1–5 μg/ml) dissociation of polysomes into 70 S monomers was not observed. Ribosomes in higher concentration of chlortetracycline (400 μg/ml) form aggregates. A decrease of Mg²⁺ to 0.1mm caused dissociation of ribosomes to two subunits and in this state none of indicated concentrations of chlortetracycline caused aggregation. The exact sedimentation values of ribosomes and ribosomal subunits were calculated from extrapolation to infinite dilution. S_20,w for monomer form was 68.8, and for ribosomal subunits 49.8 and 31.2 respectively. Ribosomal RNA sedimentates as two Schlieren peaks of 16 S and 22 S. It was found that 30 S subunits contain 15 structural proteins, while 21 proteins were resolved from 50 S subunits.     

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.     

Protein synthesis defects in temperature-sensitive mutants of Escherichia coli with altered ribosomal proteins

The ribosomes from four temperature-sensitive mutants of Escherichia coli have been examined for defects in cell-free protein synthesis. The mutants examined had alterations in ribosomal proteins S10, S15, or L22 (two strains). Ribosomes from each mutant showed a reduced activity in the translation of phage MS2 RNA at 44 degrees C and were more rapidly inactivated by heating at this temperature compared to control ribosomes. Ribosomal subunits from three of the mutants demonstrated a partial or complete inability to reassociate at 44 degrees C. 70-S ribosomes from two strains showed a reducton in messenger RNA binding. tRNA binding to the 30 S subunit was reduced in the strains with altered 30-S proteins and binding to the 50 S subunit was affected in the mutants with a change in 50 S protein L22. The relation between ribosomal protein structure and function in protein synthesis in these mutants is discussed.     

Ribosomal proteins of Bacillus subtilis vegetative and sporulating cells

Summary The ribosomal subunit proteins (30S and 50S) from vegetative and sporulating cells of Bacillus subtilis 168M were analyzed by two dimensional acrylamide gel electrophoresis. Twenty two proteins were identified in the 30S subunits and 28 proteins are detectable in the 50S subunits. The number of proteins and their electrophoretic mobility seem to remain unaltered during the sporulation process.The ribosomal proteins of a thermosensitive sporulation mutant (ts-4), isolated from stationary phase cultures, under permissive (for sporulation) and non-permissive conditions, did not show any qualitative difference in either of the subunits.The 21S precursor particles derived from log phase cell ribosomes show two different proteins, in addition to those present in the 30 S subunit. It is suggested that these two proteins either disappear or are modified during the maturation process.     

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.