Structural heterogeneity of 30S and 50S ribosomal subunits in Bacillus licheniformis

Proteins of free and polysome-derived ribosomal subunits of Bacillus licheniformis were fractionated by means of two-dimensional polyacrylamide gel electrophoresis. The free and derived 30S subunits have twelve protein components in common. Five proteins are present on the free 30S subunits only, whereas four proteins are exclusively found on the derived 30S subunits. The free and derived 50S subunits have at least twenty-eight proteins in common. Four proteins are unique for the derived and one for the free 50S subunits.     

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.     

Ribosomal proteins of Streptomyces aureofaciens producing tetracycline

Three different two-dimensional polyacrylamide gel electrophoretic systems were employed for identification of individual ribosomal proteins of Streptomyces aureofaciens. Proteins of small subunits were resolved into 21 spots. Larger ribosomal subunits contained 35 proteins. The separated ribosomal proteins from 50 S subunits were transferred on nitrocellulose membranes for immunochemical estimations. Antibodies developed against 50 S proteins of S. aureofaciens and Escherichia coli were used for identification of structural homologies between 50 S proteins of the two species. Results of the experiments indicate that about one half of the 50 S proteins of S. aureofaciens share common immunochemical determinants with corresponding proteins of 50 S subunits of E. coli. Evidence is presented that acidic ribosomal protein SL5 of large ribosomal subunits of S. aureofaciens can be assembled to E. coli P₀ cores lacking proteins L7/L12. Reconstitution of the P₀ cores with proteins SL5 or L7/L12 led to restoration of 78% activity in polyphenylalanine synthesis.     

Protein-protein proximity in the association of ribosomal subunits of Escherichia coli: crosslinking of 30 S protein S16 to 50 S proteins by glutaraldehyde or formaldehyde

The interaction of ribosomal subunits from Escherichia coli has been studied using crosslinking reagents. Radioactive ³⁵S-labeled 50 S subunits and non-radioactive 30 S subunits were allowed to reassociate to form 70 S ribosomes. The 70 S particles, containing radioactivity only in the 50 S protein moiety, were incubated with glutaraldehyde or formaldehyde. As a result of this treatment a substantial fraction of the 70 S particles did not dissociate at 1 mm-Mg²⁺. This fraction was isolated and the ribosomal proteins were extracted. The protein mixture was analyzed by the Ouchterlony double diffusion technique by using eighteen antisera prepared against single 30 S ribosomal proteins (all except those against S3, S15 and S17). As a result of the crosslinking procedure it was found that only anti-S16 co-precipitated ³⁵S-labeled 50 S protein. It is concluded that the 30 S protein S16 is at or near the site of interaction between subunits and can become crosslinked to one or more 50 S ribosomal proteins.     

Functional 70S hybrid ribosomes from blue-green algae and bacteria

Hybrid 70S ribosomes were produced by combining Anacystis nidulans and Escherichia coli 30S and 50S subunits. Both the A. nidulans 30S-E. coli 50S and E. coli 30S- A. nidulans 50S hybrids were functional in synthesizing protein when tested in a standard in vitro amino acid incorporating system. Both 70S hybrids were inhibited by streptomycin but the degree of inhibition was dependent upon the source of the 30S subunit. The ability to form functional 70S ribosomes from subunits of blue-green algae and bacteria is further evidence of the procaryotic nature of blue-greens and of the functional homology of the two protein synthesizing systems.     

Comparison of the DING protein from the archaeon Sulfolobus solfataricus with human phosphate-binding protein and Pseudomonas fluorescence DING counterparts

DING proteins represent a new group of 40 kDa-related members, ubiquitous in living organisms. The family also include the DING protein from Sulfolobus solfataricus, functionally related to poly(ADP-ribose) polymerases. Here, the archaeal protein has been compared with the human Phosphate-Binding Protein and the Pseudomonas fluorescence DING enzyme, by enzyme assays and immune cross-reactivity. Surprisingly, as the Sulfolobus enzyme, the Human and Pseudomonas proteins display poly(ADP-ribose) polymerase activity, whereas a phosphatase activity was only present in Sulfolobus and human protein, despite the conserved phosphate-binding site residues in Pseudomonas DING. All proteins were positive to anti-DING antibodies and gave a comparable pattern of anti-poly(ADP-ribose) polymerase immunoreactivity with two bands, at around 40 kDa and roughly at the double of this molecular mass. The latter signal was present in all Sulfolobus enzyme preparations and proved not due to either a contaminant or a precursor protein, but likely being a dimeric form of the 40 kDa polypeptide. The common immunological and partly enzymatic behavior linking human, Pseudomonas and Sulfolobus DING proteins, makes the archaeal protein an important model system to investigate DING protein function and evolution within the cell.

     

Proteomic analysis of secreted membrane vesicles of archaeal Sulfolobus species reveals the presence of endosome sorting complex components

The crenarchaea Sulfolobus acidocaldarius, S. solfataricus and S. tokodaii, release membrane vesicles into the medium. These membrane vesicles consist of tetraether lipids and are coated with an S-layer. A proteomic analysis reveals the presence of proteins homologous to subunits of the eukaryotic endosomal sorting complex required for transport (ESCRT). Immunodetection of one of these homologs suggest a cell surface localization in intact cells. These data suggest that the membrane vesicles in Sulfolobus sp. emerge from a specific budding process with similarity to the endosomal sorting pathway.     

The protein composition of Mycoplasma capricolum

Summary The whole cell proteins and the ribosomal proteins of Mycoplasma capricolum ATCC 27343 have been analyzed by two-dimensional polyacrylamide gel electrophoresis. The M. capricolum cell is relatively rich in basic proteins. The number of total protein spots detected was approximately 355, which is less than one-third of that of Escherichia coli or Bacillus subtilis. In contrast, the number (30 and 20 protein species have been found to be present in the 50S and 30S ribosomal subunits, respectively) and the size of the ribosomal proteins in the M. capricolum do not seem to be significantly different from those of typical eubacteria.     

Proteins occurring at, or near, the subunit interface of E. coli ribosomes

Summary The identification of ribosomal proteins that occur at, or near, the subunit interface of the 30S and 50S subunits in the E. coli 70S ribosome was attempted by studying the effect of antibodies on the Mg⁺⁺ dependent dissociation-association equilibrium of 70S ribosomes. Dissociated ribosomes were mixed with monovalent fragments of IgG antibodies (Fab's) specific for each ribosomal protein and then reassociated into intact 70S particles. Various degrees of inhibition of this reassociation were observed for proteins S9, S11, S12, S14, S20, L1, L6, L14, L15, L19, L20, L23, L26 and L27. A small amount of aggregation of 50S subunits was caused by IgG's specific for the proteins S9, S11, S12, S14 and S20 and purified 50S subunits. It was inferred that the presence of small amounts of these proteins on 50S subunits was compatible with their presence at the subunit interface. Finally, the capacity of proteins S11 and S12 to bind to 23S RNA was demonstrated.Paper No. 84 on Ribosomal Proteins. Preceding paper is by Rahmsdorf et al., Molec. gen. Genet. 127, 259–271 (1973).     

Comparison of ribosomal proteins of chloroplast from spinach and of E. coli

Summary A comparison of ribosomal proteins from Escherichia coli and from chloroplasts of Spinach was made using two separate methods: electrophoretic migration and immunochemical cross-reaction between blotted E. coli ribosomal proteins and chloroplast ribosomal subunits antisera. It is shown that L2 from E. coli (E-12) and L4 from chloroplasts (CS-L4) comigrated and that E-L4 immunologically cross-reacted with the isolated CS-L4 antibody. Co-migration was observed for three additional couples of 50S ribosomal proteins. It is also shown that at least one 30S E. coli ribosomal protein immuno-cross reacted with a 30S chloroplast antiserum and that three couples of 30S ribosomal proteins comigrated.     

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.     

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.     

The subunit interface of the Escherichia coli ribosome. Crosslinking of 30 S protein S9 to proteins of the 50 S subunit.

Purified 50 S ribosomal subunits were found to contain significant amounts of protein coincident with the 30 S proteins S9 and/or S11 on two-dimensional polyacrylamide/urea electropherographs. Peptide mapping established that the protein was largely S9 with smaller amounts of S11. Proteins S5 and L6 were nearly coincident on the two-dimensional polyacrylamide/urea electropherographs. Peptide maps of material from the L6 spot obtained from purified 50 S subunits showed the presence of significant amounts of the peptides corresponding to S5. Experiments in which ³⁵S-labelled 30 S subunits and non-radioactive 50 S subunits were reassociated to form 70 S ribosomes showed that some radioactive 30 S protein was transferred to the 50 S subunit. Most of the transferred radioactivity was associated with two proteins, S9 and S5. Sulfhydryl groups were added to the 50 S subunit by amidination with 2-iminothiolane (methyl 4-mercaptobutyrimidate). These were oxidized to form disulfide linkages, some of which crosslinked different proteins of the intact 50 S ribosomal subunit. Protein dimers were partially fractionated by sequential salt extraction and then by electrophoresis of each fraction in polyacrylamide gels containing urea. Slices of the gel were analysed by two-dimensional polyacrylamide/sodium dodecyl sulfate diagonal gel electrophoresis. Final identification of the constituent proteins in each dimer by two-dimensional polyacrylamide/urea gel electrophoresis showed that 50 S proteins L5 and L27 were crosslinked to S9. The evidence suggests that proteins S5, S9, S11, L5 and L27 are located at the interface region of the 70 S ribosome.     

Functional heterogeneity of the 30S ribosomal subunit of Escherichia coli. II. Effect of S21 on initiation

Summary Native 30S ribosomal subunits fromEscherichia coli are deficient in fractional protein S21, which is present on the monosome and polysome-derived 30S subunits. The presence of S21 prevents the binding of Fmet-tRNA if and only if 50S subunits are present. In contrast, proteins S2, S3 and S14 stimulate the binding of Fmet-tRNA. These results have been used to rationalize other data concerning the mechanism of Fmet-tRNA binding by ribosomes. In addition, the present data indicate that the 30S ribosomal subunits are heterogeneousin vivo as well asin vitro.Dedicated to Professor H. Veldstra on the occasion of his retirement from the chair of biochemistry of the University of Leiden.     

Isolation of active 30S and 50S ribosomal subunits from a psychrotrophic bacterium

Ribosomes from the psychrotroph,Bacillus insolitus, were successfully dissociated into 30S and 50S ribosomal subunits, which were active in carrying out in vitro protein synthesis, measured by the poly U-directed incorporation of¹⁴C-l-phenylalanine into polyphenylalanine. As opposed to the undissociated ribosomes, which are heat sensitive (30°C), the dissociated ribosomes were not thermally sensitive. The heat-sensitive component(s) was found to be removed from the ribosomes during dissociation into 30S and 50S ribosomal subunits; when added back to the ribosomal subunits, heat sensitivity was conferred.     

The RimP Protein Is Important for Maturation of the 30S Ribosomal Subunit

The in vivo assembly of ribosomal subunits requires assistance by auxiliary proteins that are not part of mature ribosomes. More such assembly proteins have been identified for the assembly of the 50S than for the 30S ribosomal subunit. Here, we show that the RimP protein (formerly YhbC or P15a) is important for the maturation of the 30S subunit. A rimP deletion (ΔrimP135) mutant in Escherichia coli showed a temperature-sensitive growth phenotype as demonstrated by a 1.2-, 1.5-, and 2.5-fold lower growth rate at 30, 37, and 44 °C, respectively, compared to a wild-type strain. The mutant had a reduced amount of 70S ribosomes engaged in translation and showed a corresponding increase in the amount of free ribosomal subunits. In addition, the mutant showed a lower ratio of free 30S to 50S subunits as well as an accumulation of immature 16S rRNA compared to a wild-type strain, indicating a deficiency in the maturation of the 30S subunit. All of these effects were more pronounced at higher temperatures. RimP was found to be associated with free 30S subunits but not with free 50S subunits or with 70S ribosomes. The slow growth of the rimP deletion mutant was not suppressed by increased expression of any other known 30S maturation factor.     

Comparative electron microscopic study on the location of ribosomal proteins S3 and S7 on the surface of the E. coli 30S subunit using monoclonal and conventional antibody

Summary Mice were immunised with 30S subunits from E. coli and their spleen cells were fused with myeloma cells. From this fusion two monoclonal antibodies were obtained, one of which was shown to be specific for ribosomal protein S3, the other for ribosomal protein S7. The two monoclonal antibodies formed stable complexes with intact 30S subunits and were therefore used for the three-dimensional localisation of ribosomal proteins S3 and S7 on the surface of the E. coli small subunit by immuno electron microscopy. The antibody binding sites determined with the two monoclonal antibodies were found to lie in the same area as those obtained with conventional antibodies. Both proteins S3 and S7 are located on the head of the 30S subunit, close to the one-third/two-thirds partition. Protein S3 is located just above the small lobe, whereas protein S7 is located on the side of the large lobe.     

Comparative study of the extracellular proteome of Sulfolobus species reveals limited secretion

Although a large number of potentially secreted proteins can be predicted on the basis of genomic distribution of signal sequence-bearing proteins, protein secretion in Archaea has barely been studied. A proteomic inventory and comparison of the growth medium proteins in three hyperthermoacidophiles, i.e., Sulfolobus solfataricus, S. acidocaldarius and S. tokodaii, indicates that only few proteins are freely secreted into the growth medium and that the majority originates from cell envelope bound forms. In S. acidocaldarius both cell-associated and secreted α-amylase activities are detected. Inactivation of the amyA gene resulted in a complete loss of activity, suggesting that the same protein is responsible for the a-amylase activity at both locations. It is concluded that protein secretion in Sulfolobus is a limited process, and it is suggested that the S-layer may act as a barrier for the free diffusion of folded proteins into the medium.     

A 50S Ribosomal Subunit Precursor Particle Is a Substrate for the ErmC Methyltransferase in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> Cells

Macrolide antibiotics like erythromycin can induce the synthesis of a specific 23S rRNA methyltransferase which confers resistance to cells containing the erm gene. Erythromycin inhibits both protein synthesis and the formation of 50S subunits in bacterial cells. We have tested the idea that the 50S precursor particle that accumulates in antibiotic-treated Staphylococcus aureus cells is a substrate for the methyltransferase enzyme. Pulse-chase labeling studies were conducted to examine the rates of ribosomal subunit formation in control and erythromycin-induced cells. Erythromycin binding to 50S subunits was examined under the same conditions. The rate of 50S subunit formation was reduced for up to 30 min after antibiotic addition, and erythromycin binding was substantial at this time. A nuclease protection assay was used to examine the methylation of adenine 2085 in 23S rRNA after induction. A methyl-labeled protected RNA sequence was found to appear in cells 30 min after induction. This protected sequence was found in both 50S subunits and in a subunit precursor particle sedimenting at about 30S in sucrose gradients. 23S rRNA isolated from 50S subunits of cells could be labeled by a ribosome-associated methlytransferase activity, with ³H-S-adenosylmethionine as a substrate. 50S subunits were not a substrate for the enzyme, but the 30S gradient region from erythromycin-treated cells contained a substrate for this activity. These findings are consistent with a model that suggests that antibiotic inhibition of 50S formation leads to the accumulation of a precursor whose 23S rRNA becomes methylated by the induced enzyme. The methylated rRNA will preclude erythromycin binding; thus, assembly of the particle and translation become insensitive to the inhibitory effects of the drug. Received: 21 June 2002 / Accepted: 21 August 2002     

Translational system of the hydrogen-oxidizing bacterium Alcaligenes eutrophus

Some structural and functional properties of ribosomes from the hydrogen-oxidizing bacterium Alcaligenes eutrophus were studied in order to investigate the background of expression of genetic information at the translational level. Ribosomal proteins from 30S subunits of A. eutrophus H16 were separated by two-dimensional gel electrophoresis into 21 spots, those from 50S subunits into 32 spots. While electrophoretic mobilities of several ribosomal proteins differed markedly from those of Escherichia coli, proteins sharing common immunological determinants with E. coli ribosomal proteins S1 and L7/L12 were found in A. eutrophus. Shifting from heterotrophic to autotrophic conditions of growth had no influence on the ribosomal protein pattern. Ribosomes of A. eutrophus had similar requirements for Mg²⁺ and poly(U) concentrations for optimum polyphenylalanine synthesis as those of E. coli. Protein synthesis elongation factors Tu from A. eutrophus and E. coli were immunologically similar. Efficiency of the A. eutrophus polyphenylalanine-synthesizing system was comparable to that of an analogous system derived from E. coli. This suggests that A. eutrophus could be employed for efficient expression of recombinant DNA.