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.     

Stoichiometric measurements of 30S and 50S ribosomal proteins from Escherichia coli

Summary Stoichiometric investigations were made for all 21 proteins of the 30S subunit and for 31 proteins of the 50S subunit. While the results for the 30S proteins strongly suggest the existence of two different stoichiometric classes, the so-called fractional (F) and unit (U) proteins with average molar amounts of 0.1–0.5 and 0.8–1.2 per mole 30S respectively, at least one further group is present in isolated 50S particles. Therefore 50S proteins may be subdivided in the following way: a) 9–10 proteins with average molar amounts of 0.2–0.6 (F-proteins), b) 15–16 proteins with 0.8–1.2 copies per 50S particle (U-proteins), c) at least one protein with an average value of 1.8–2.2 copies, the repeated (R) proteins, d) a group of 7 proteins with 1.4–1.7 copies per particle; they can be named fractional repeated (FR) proteins. These findings indicate that 50S ribosomal particles show a more differentiated degree of structural heterogeneity at least in vitro than has been shown for 30S subunits.Paper No. 35 on Ribosomal Proteins. Preceding paper is by E. Deusser and H. G. Wittmann, Nature 238, 269 (1972).     

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.     

Immunoelectron microscopic studies on the location of ribosomal proteins on the surface of the 40S ribosomal subunit from rat liver

Seven ribosomal proteins have been localized by means of immunoelectron microscopy on the surface of the 40S ribosomal subunit from rat liver using monospecific antibodies. The location of ribosomal proteins S13/16, S19, and S24 is described for the first time, and that of ribosomal proteins S2, S3, S3a, and S7, which has been published previously on the basis of experiments performed with less well characterized antibody preparations [Lutsch et al., Mol. Gen. Genet. 176, 281-291 (1979) and Biomed. Biochim. Acta 42, 705-723 (1983)], is corrected in this paper. The results are discussed with respect to the involvement of these proteins in functional sites of the 40S ribosomal subunit.     

Revised location of the Escherichia coli gene coding for ribosomal proteins S2

Summary P1 transduction studies of a mutation of Escherichia coli affecting the rpsB gene previously suggested the map order popC-rpsB-dapD. Biochemical analysis of transductants has now revealed that the rpsB gene lies in fact to the right hand (tsf) side of the dapD marker.     

Epitope mapping of monoclonal antibodies toEscherichia coli ribosomal protein S3

The antigenic structure ofEscherichia coli ribosomal protein S3 has been investigated by use of monoclonal antibodies. Six S3-specific monoclonal antibodies secreted by mouse hybridomas have been identified by immunoblotting of two-dimensional ribosomal protein separation gels. By using a competitive enzyme-linked immunosorbent assay, we have divided these monoclonal antibodies into three mutual inhibition groups, members of which are directed to three distinct regions of the S3 molecule. The independence of these monoclonal antibody-defined regions was confirmed by the failure of pairs of monoclonal antibodies from two inhibition groups to block the binding of biotinylated monoclonal antibodies of the third group. To determine the regions recognized by these monoclonal antibodies, chemically cleaved S3 peptides were fractionated by gel filtration and reverse-phase high-performance liquid chromatography. The fractionated peptides were coated on plates and examined for specific interaction with monoclonal antibody by enzyme immunoassay. In this manner, two epitopes have been mapped at the ends of the S3 molecule: one, in the last 22 residues, is recognized by three monoclonal antibodies; and the second, in the first 21 residues, is defined by two monoclonal antibodies. The third S3 epitope, recognized by a single monoclonal antibody, has been localized in a central segment of about 90 residues by gel electrophoresis and immunoblotting. These epitope-mapped monoclonal antibodies are valuable probes for studying S3 structurein situ.     

Electron microscopic localization of two proteins on the surface of the 50 S ribosomal subunit of Escherichia coli using specific antibody markers

A method to localize individual proteins on the surface of the ribosomal subunit was developed. The method uses specific immunoglobulins G against proteins under examination. The antibodies are combined with the ribosomes to give rise to ribosome dimers linked by the bivalent antibody. The Fab arm of the Y shaped antibody points to the position of the protein the antibody was prepared against.Using this method, two ribosomal proteins (L1, L19)³were located on two defined shapes of the 50 S ribosomal subunit.     

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.     

A role for proteins S3 and S14 in the 30 S ribosomal subunit

Small ribosomal subunits prepared by the method of Kirillov et al. (Kirillov, S. V., Makhno, V. I., Peshin, N. N., and Semenkov, Yu. P. (1986) Nucleic Acids Res. 5, 4305-4315) are active but fail to reconstitute. The inability to reconstitute is due to a deficiency in proteins S3 and S14. Supplementation of the protein component with pure S3 and S14 leads to an enhancement of the activity of the reconstituted product. Our results provide evidence that these two proteins are involved in assembly but may not be required once the 30 S subunit has been properly assembled.     

The accessibility of proteins of the Escherichia coli 30S ribosomal subunit to antibody binding

Summary The accessibility of each of the proteins on the E. coli 30S ribosomal subunit was established by investigating whether or not immunoglobulins (IgG's) and their monovalent papain fragments (Fab's), specific for each of the 21 single ribosomal proteins, bind to the 30S subunit. The interpretation of the results of five different experimental approaches, namely Ouchterlony double diffusion and immunological sandwich methods, sucrose gradient and analytical ultracentrifugation, and functional inhibition tests, indicate that all 21 proteins of the 30S subunit have determinants available for antibody binding. There were quantitative differences between the degree of accessibility of the different ribosomal proteins. An attempt was made to correlate the results with the protein stoichiometric data of the small subunit proteins.     

The topography of ribosomal proteins on the surface of the 30S subunit of Escherichia coli

Eight ribosomal proteins, S6, S10, S11, S15, S16, S18, S19 and S21 have been localized on the surface of the 30S subunit from Escherichia coli by immuno electron microscopy. The specificity of the antibody binding sites was demonstrated by stringent absorption experiments. In addition we have reinvestigated and refined the locations of proteins S5, S13 and S14 on the ribosomal surface which had previously been localized in our laboratory (Tischendorf et al., Mol. Gen. Genet. 134, 209-223, 1974). Thus altogether 16 out of the 21 ribosomal proteins of the small subunit from E. coli have been mapped in our laboratory.     

Site-specific properties of Tn7 transposition into the E. coli Chromosome

Summary A study has been made of the insertional properties of transposon Tn7, a 14 kilobase transposable element encoding resistances to trimethoprim, streptomycin and specitinomycin. It has previously been shown that Tn7 transposes at a low frequency and with low specificity into multiple sites in large transmissible plasmids. However, Tn7 transposes with extrame specificity and at high efficiency into the E. coli chromosome. In all cases we have studied, insertion of Tn7 into the chromosome has occurred at a unique site and with a unique orientation. A combination of genetic and biochemical techniques have been used to precisely locate this site on the E. coli chromosome to minute 82 on the linkage map between markers glmS and uncA.To investigate the nature of this highly specific transpositional event, a small region of the E. coli chromosome that includes the unique site, was cloned into the plasmid vector pBR322. Subsequently a lkb restriction fragment, including the Tn7 insertion site, was sub-cloned from this plasmid into the plasmid pACYC184. We show that Tn7 transposes into both these plasmid recombinants with the frequency and specificity characteristie of the E. coli chromosome.     

Functional homology between E. coli ribosomal protein L11 and B. megaterium protein BM-L11

Summary Ribosomes from the thiostrepton-resistant mutant MJ1 of Bacillus megaterium completely lack a protein designated BM-L11. When assayed in vitro, such ribosomes show an impaired ability to hydrolyse GTP in the presence of the elongation factor EF-G and are unable to support the synthesis of (p)ppGpp in response to the stringent factor. Restoration of both these activities can be achieved by re-addition of either protein BM-L11 or its serological homologue from Escherichia coli, protein L11, implying that these two proteins are related functionally as well as immunologically.     

Isolation of the mini insertions IS6 and IS7 of E. coli

Summary The mini IS elements IS6 and IS7 have been detected in constitutive gal ⁺ revertants of galOP-308::IS2 (I), in which the expression of the gal operon is turned off by IS2 in orientation I. Both, IS6 and IS7, are integrated into IS2 proximal to the gal structural genes. IS6 is 115 base pairs long and causes 50% constitutive expression of the gal genes. IS7 is only 65 base pairs long and the gal operon is expressed 20% constitutively compared to the gal ⁺ wild type operon. Both IS6 and IS7 are excised frequently, in the absence of selective pressure. These findings are discussed with respect to the evolution of gene expression.