Localization of ribosomal protein S2 on the surface of the 30S subunit from Escherichia coli, using monoclonal antibodies.

Protein S2 has been localized on the surface of the 30S subunit of Escherichia coli by immuno-electron microscopy. The antibody was obtained from a fusion of myeloma cells with spleen cells of mice, which had been immunized with intact 30S ribosomal subunits of E. coli. The binding site of the antibody was on the head of the small subunit, just above the small lobe, in the region where protein S3 has also been localized. S2 is the first ribosomal protein to have been mapped exclusively with monoclonal antibody.     

Differential assembly of 16S rRNA domains during 30S subunit formation

Rapid and accurate assembly of the ribosomal subunits, which are responsible for protein synthesis, is required to sustain cell growth. Our best understanding of the interaction of 30S ribosomal subunit components (16S ribosomal RNA [rRNA] and 20 ribosomal proteins [r-proteins]) comes from in vitro work using Escherichia coli ribosomal components. However, detailed information regarding the essential elements involved in the assembly of 30S subunits still remains elusive. Here, we defined a set of rRNA nucleotides that are critical for the assembly of the small ribosomal subunit in E. coli. Using an RNA modification interference approach, we identified 54 nucleotides in 16S rRNA whose modification prevents the formation of a functional small ribosomal subunit. The majority of these nucleotides are located in the head and interdomain junction of the 30S subunit, suggesting that these regions are critical for small subunit assembly. In vivo analysis of specific identified sites, using engineered mutations in 16S rRNA, revealed defective protein synthesis capability, aberrant polysome profiles, and abnormal 16S rRNA processing, indicating the importance of these residues in vivo. These studies reveal that specific segments of 16S rRNA are more critical for small subunit assembly than others, and suggest a hierarchy of importance.     

Location of protein S4 on the small ribosomal subunit of E. coli and B. stearothermophilus with protein- and hapten-specific antibodies

Summary In spite of considerable effort there is still serious disagreement in the literature about the question of whether epitopes of ribosomal protein S4 are accessible for antibody binding on the intact small ribosomal subunit. We have attempted to resolve this issue using three independent approaches: (i) a re-investigation of the exposure and the location of epitopes of ribosomal protein S4 on the surface of the 30S subunit and 30S core particles of the E. coli ribosome, including rigorous controls of antibody specificity, (ii) a similar investigation of protein S4 from Bacillus stearothermophilus and (iii) the labelling of residue Cys-31 of E. coli S4 with a fluorescein derivative the accessibility of which towards a fluorescein-specific antibody was demonstrated directly by fluorimetry. In each of the three cases the antigen (E. coli S4, B. stearothermophilus S4 or fluorescein) was found to reside on the small lobe.     

Interaction of the release factor with the Escherichia coli ribosome: inhibition of the 30S subunit domain by specific antibodies

A domain of the 30S subunit of the Escherichia coli ribosome is in close contact with the release factor when it binds to the 70S particle during the termination of protein biosynthesis. This has been characterised using antibodies specific for the individual proteins of the small ribosomal subunit. Most antibodies do not affect the release factor-mediated reactions but those against S3, S4, S5 and S10 are inhibitory. These proteins are clustered on the lower head and the upper part of the small lobe of the subunit. The regions of these features which are near the interface between the two subunits in the 70S ribosome are known to be close to the base of the stalk of the 50S subunit.     

Both ends of Escherichia coli ribosomal protein S13 are immunochemically accessible in situ.

To investigate the structure ofEscherichia coli ribosomal protein S13 in 30S ribosomal subunits, we have previously generated 22 S13 specific monoclonal antibodies and mapped their specific epitopes to the S13 sequence. The availability of these S13 epitopesin situ has been further examined by incubating these monoclonal antibodies with 30S ribosomal subunits and analyzing formation of monoclonal antibody-linked ribosome dimers by sucrose gradients centrifugation. We have found that none of the 22 monoclonal antibodies makes ribosome dimers individually as do typical antisera. However, one monoclonal antibody, designated AS13-MAb 2, reacts with 30S ribosomal subunits to form immunocomplexes sedimenting faster than subunit monomers. When AS13-MAb 2 is paired with any one of three monoclonal antibodies directed to the S13 C-terminal epitopes, dimer formation is observed. Other pairs of monoclonal antibodies directed to distinct S13 epitopes have been tested similarly for dimer formation. Monoclonal antibody AS13-MAb 22, directed to the N-terminal region of 22 residues, also causes subunits to form typical dimers, but only if paired with one of the three monoclonal antibodies directed to the S13 C-terminal region. The close proximity of the epitopes recognized by AS13-MAbs 2 and 22 has been established by the mutual competition between the antibodies binding to intact 30S subunits. These results corroborate our previous observation, using polyclonal antibodies, that S13 has more than one epitope exposed on 30S subunits. Our finding that sequences on both ends of the S13 molecule are immunochemically accessible provides information about the molecular organization of S13in situ.     

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.     

Three-dimensional localization of the NH2- and carboxyl-terminal domain of ribosomal protein S1 on the surface of the 30 S subunit from Escherichia coli.

Antibodies were raised against Escherichia coli ribosomal protein S1 and its NH2- and COOH-terminal fragments, and their specificity was demonstrated by a variety of immunological techniques. These antibodies were then used to investigate the location of protein S1 and its NH2- and COOH-terminal domains on the surface of the 30 S ribosomal subunit by immunoelectron microscopy. In order to prevent dissociation of the protein during the experiments, S1 was cross-linked to 30 S subunits with dithiobis(succinimidyl-propionate); cross-linking yield was 100%. Epitopes of the NH2-terminal domain of S1 were localized at the large lobe of the 30 S ribosomal subunit, close to the one-third/two-thirds partition on the side which in the 70 S ribosome faces the cytoplasm. Experiments with monovalent Fab fragments specific for the COOH-terminal part of S1 provide evidence that the COOH-terminal domain forms an elongated structure extending at least 10 nm from the large lobe of the small subunit into the cytoplasmic space.     

Late steps in the assembly of 30 S ribosomal proteins in vivo in a spectinomycin-resistant mutant of Escherichia coli.

The late steps in ribosome assembly in vivo were studied by characterizing mutations which suppress the cold-sensitivity of a spectinomycin-resistant mutant of Escherichia coli. The results obtained indicated that the cold-sensitivity could be relieved by secondary alterations in either the S2, S3 or S5 protein of the 30 S ribosomal subunit. The gene controlling the alteration of S2 protein was closely linked to the polC gene located at about 3.5 minutes on the genetic map of E. coli, whereas S3 and S5 suppressor genes were linked to the str-spc region at 64 minutes. A possible model in which the S2, S3 and S5 proteins constitute a sub-assembly pathway in the assembly of 30 S subunits in vivo is discussed.     

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.     

Ribosomal proteins S3, S6, S8 and S10 of Escherichia coli localized on the external surface of the small subunit by immune electron microscopy

The three-dimensional locations of Escherichia coli ribosomal proteins S3, 86, S8 and S10 on the surface of the small subunit were determined by immune electron microscopy.All four proteins are located on the “external surface” of the small subunit; i.e. on the side of the subunit in contact with the cytosol in the 70 S ribosome. Proteins S3, S6, S8 and S10 map at single sites, although the S3 site is extended approximately 40Å along the long axis of the subunit. S8 is located near the base of the cleft separating the platform from the upper one-third or head; protein S10 is located in the head, near the site previously mapped for S14; S3 extends from the level of the constriction to near the top of the head in the vicinity of S10; and S6 is located on the platform of the small subunit near the site previously mapped for S11.The locations of these proteins correlate well with other information on their spatial relationships obtained from assembly interactions, neutron diffraction, crosslinking and protein associations.     

Both ends ofEscherichia coli ribosomal protein S13 are immunochemically accessiblein situ

To investigate the structure ofEscherichia coli ribosomal protein S13 in 30S ribosomal subunits, we have previously generated 22 S13 specific monoclonal antibodies and mapped their specific epitopes to the S13 sequence. The availability of these S13 epitopesin situ has been further examined by incubating these monoclonal antibodies with 30S ribosomal subunits and analyzing formation of monoclonal antibody-linked ribosome dimers by sucrose gradients centrifugation. We have found that none of the 22 monoclonal antibodies makes ribosome dimers individually as do typical antisera. However, one monoclonal antibody, designated AS13-MAb 2, reacts with 30S ribosomal subunits to form immunocomplexes sedimenting faster than subunit monomers. When AS13-MAb 2 is paired with any one of three monoclonal antibodies directed to the S13 C-terminal epitopes, dimer formation is observed. Other pairs of monoclonal antibodies directed to distinct S13 epitopes have been tested similarly for dimer formation. Monoclonal antibody AS13-MAb 22, directed to the N-terminal region of 22 residues, also causes subunits to form typical dimers, but only if paired with one of the three monoclonal antibodies directed to the S13 C-terminal region. The close proximity of the epitopes recognized by AS13-MAbs 2 and 22 has been established by the mutual competition between the antibodies binding to intact 30S subunits. These results corroborate our previous observation, using polyclonal antibodies, that S13 has more than one epitope exposed on 30S subunits. Our finding that sequences on both ends of the S13 molecule are immunochemically accessible provides information about the molecular organization of S13in situ.     

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.     

Analysis of kethoxal bound to ribosomal proteins from Escherichia coli 70S reacted ribosomes

To investigate ribosome topography and possible function, 70S ribosomes of $\text{Escherichia coli}$ were reacted with the dicarbonyl compound kethoxal. Ribosomal protein was extracted after reaction, and through two dimensional gel electrophoresis, the reactive proteins of the two subunits were identified. From the 30S subunit, the most reacted proteins were S2, S3, S4, S5 and S7 and from the 50S subunit, L1, L5, L16, L17, L18 and L27. The results with kethoxal are compared with other modifiers of ribosomal proteins.     

E. coli ribosomal proteins are cross reactive with antibody prepared against Chlamydomonas reinhardi chloroplast ribosomal subunit

Summary Antisera prepared against purified Chlamydomonas reinhardi small chloroplast ribosomal subunit, judged homogenous by sucrose gradient velocity sedimentation and RNA gel electrophoresis was immunologically cross reactive with E. coli ribosomal proteins. The results of three different experimental approaches, namely Ouchterlony double diffusion, sucrose gradient velocity sedimentation and two dimensional crossed immunoelectrophoresis indicate that both E. coli ribosomal subunits and the chloroplast large ribosomal subunit contain proteins which show antigenic similarity to the chloroplast small ribosomal subunit proteins. However, cytoplasmic ribosomal subunits did not contain proteins which were cross reactive with immune antisera.     

Purification and characterization of 30S ribosomal proteins from Bacillus subtilis: correlation to Escherichia coli 30S proteins

Summary Twenty proteins were isolated from the 30S ribosomal subunits of Bacillus subtilis and their amino acid compositions and amino-terminal amino acid sequences were determined. These results were compared with the data of Escherichia coli 30S ribosomal proteins and the structural correspondence of individual ribosomal proteins has been established between B. subtilis and E. coli.Post-translational modifications of amino-terminal amino acids of the ribosomal proteins which have been found in E. coli are almost absent in B. subtilis with the exception of acetylated forms of S9.     

Structure of ribosomes and ribosomal subunits of Drosophila

Summary The ultrastructure of Drosophila melanogaster cytoplasmic ribosomal subunits and monomers have been examined by electron microscopy. The Drosophila ribosomal structures are compared to those determined for other eucaryotes and E. coli. Negatively contrasted images of 60S subunits are seen in the most frequent view to be approximately round particles about 280 Å in diameter. About 35% of the particles present a single prominent projection which we call the 60S peak. The peak emanates from a flattened region of the 60S subunit. The maximum observed length of the 60S peak is approximately 90Å. The Drosophila 60S peak is highly reminiscent of the E. coli L7/L12 stalk. The Drosophila 40S subunit is an elongated, slightly bent particle which measures 280×170×160 Å. It bears a strong resemblance to small ribosomal subunits of other eucaryotes and is strikingly similar to the E. coli 30S subunit. Micrographs of 80S monomeric ribosomes show the long axis of the 40S to be parallel and in apparent contact with the flattened region of 60S subunit. The 60S peak appears to bisect the long axis of the 40S subunit. The 40S subunit seems to be oriented in the monomeric ribosome so that the 40S projection is toward the body of the large subunit. Comparison of our data with similar studies in different organisms indicates that the eucaryotic large ribosomal subunits exhibit morphological heterogeneity while the small subunits remain remarkably similar.     

The functional and structural homology of ribosomal protein S7 of E. coli strains K and MRE600

Summary The 30S ribosomal protein S7 purified from E. coli MRE600 displaces specifically and stoichiometrically the endogenous K-S7 protein when it is added to a reconstitution system containing total K strain 30S protein and 16S RNA. The S7 proteins from the two strains have been shown to contain a group of common trypic peptides and to crossreact immunologically. Therefore, the 30S ribosomal protein S7 from E. coli K strain and MRE600 are functionally and structurally homologous despite differences in amino acid composition, molecular weight and electrophoretic mobility.     

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).     

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.     

Binding of erythromycin to the 50S ribosomal subunit is affected by alterations in the 30S ribosomal subunit

Summary Expression of resistance to erythromycin in Escherichia coli, caused by an altered L4 protein in the 50S ribosomal subunit, can be masked when two additional ribosomal mutations affecting the 30S proteins S5 and S12 are introduced into the strain (Saltzman, Brown, and Apirion, 1974). Ribosomes from such strains bind erythromycin to the same extent as ribosomes from erythromycin sensitive parental strains (Apirion and Saltzman, 1974).Among mutants isolated for the reappearance of erythromycin resistance, kasugamycin resistant mutants were found. One such mutant was analysed and found to be due to undermethylation of the rRNA. The ribosomes of this strain do not bind erythromycin, thus there is a complete correlation between phenotype of cells with respect to erythromycin resistance and binding of erythromycin to ribosomes.Furthermore, by separating the ribosomal subunits we showed that 50S ribosomes bind or do not bind erythromycin according to their L4 protein; 50S with normal L4 bind and 50S with altered L4 do not bind erythromycin. However, the 30s ribosomes with altered S5 and S12 can restore binding in resistant 50S ribosomes while the 30S ribosomes in which the rRNA also became undermethylated did not allow erythromycin binding to occur.Thus, evidence for an intimate functional relationship between 30S and 50S ribosomal elements in the function of the ribosome could be demonstrated. These functional interrelationships concerns four ribosomal components, two proteins from the 30S ribosomal subunit, S5, and S12, one protein from the 50S subunit L4, and 16S rRNA.