Marina Stöffler-Meilicke

Ribosomes in thiostrepton-resistant mutants of Bacillus megaterium lacking a single 50 S subunit protein.

Abstract A protein required for the binding of thiostrepton to ribosomes of Bacillus megaterium has been purified and further characterized by immunological techniques. This protein, which does not bind the drug off the ribosome, is serologically-homologous to Escherichia coli ribosomal protein L11 and is designated BM-L11. Ribosomes from certain thiostrepton-resistant mutants of B. megaterium appear to be totally devoid of protein BM-L11 as judged by modified immunoelectrophoresis. Such ribosomes are significantly less sensitive than those from wild-type organisms to the action of thiostrepton in vitro but retain substantial protein synthetic activity. Re-addition of protein BM-L11 to ribosomes from the mutants restores them to wild-type levels of activity and thiostrepton sensitivity. Thus ribosomal protein BM-L11 is involved not only in binding thiostrepton but also in determining the thiostrepton phenotype.

Characterisation of a mutant from Escherichia coli lacking protein L15 and localisation of protein L15 by immuno-electron microscopy

SummaryTwo mutants lacking protein L15 from the ribosome as determined by two dimensional gels were investigated using a number of different immunological methods. One strain was found to possess several protein L15 moieties which differed in net charge and in size. The other showed no evidence of L15 cross-reacting material (CRM) on the ribosome or in the supernatant. Ribosomes of this strain were used as a control in the process of the localisation of protein L15 on the surface of the large subunit of Escherichia coli ribosomes. Antigenic determinants mapped in the angle between the central protuberance and the L1 protuberance. Protein L15 has been assigned a central role in the large subunit in vitro assembly map, in peptidyltransferase activity and in the binding of erythromycin, so the significance of a mutant lacking this protein is discussed.

Mutants of Escherichia coli lacking ribosomal protein L1

Two independently isolated mutants of Escherichia coli, RD19 and MV17-10, that appeared to lack protein L1 on their ribosomes, as determined by two-dimensional gels, were subjected to a battery of immunological tests to find if L1 was indeed lacking. The tests involved Ouchterlony double diffusion, modified immunoelectrophoresis, dimer formation on sucrose gradients, and affinity chromatography. By all these criteria, protein L1 was missing from the ribosome in these mutants. Nor was any L1 cross-reacting material detectable in the supernatant. There was, however, a specific two- to fivefold increase in concentrations of protein L11 in the supernatants of the mutants, which was evidence that protein L1 acts as a feedback inhibitor of expression of the operon coding for the genes for proteins L11 and L1. Electron micrographs of ribosomes obtained from these mutants were indistinguishable from those of wild-type strains. 50 S ribosomal subunits from mutants RD19 and MV17-10 were reconstituted with purified L1 from wild-type and investigated by immunoelectron microscopy. The three-dimensional location of ribosomal protein L1 on the surface of the large subunit was determined. L1 is located on the wider lateral protuberance of the 50 S subunit. The position of protein L1 in 50 S subunits reconstituted from mutants RD19 and MV17-10 was indistinguishable from the position in subunits from wild-type.

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

SummaryMice 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.

Immunoelectron microscopy of ribosomes carrying a fluorescence label in a defined position: Location of proteins S17 and L6 in the ribosome of Escherichia coli

By coupling fluorescein to a defined amino acid of a single ribosomal protein and incorporating this protein into the ribosome, we have obtained ribosomes labelled at a single, defined position. A fluorescein‐specific antibody preparation was used to locate the fluorescein residues bound to the two cysteines at positions 58 and 63 of protein S17 and to the cysteine at position 86 of protein L6. This study demonstrates the advantages which accrue from the combination of electron microscopy and fluorimetry.

Localization of the 3' end of 16S rRNA in Escherichia coli 30S ribosomal subunits by immuno electron microscopy.

SummaryThe location of the 3′ end of 16S rRNA in E. coli 30S ribosomal subunits has been determined by immuno electron microscopy. The 3′ terminal adenosine of isolated 16S rRNA was oxidized with sodium periodate and reacted with N-γ-(2,4-dinitrophenyl) aminobutyric acid hydrazide. Functionally active 30S subunits were reconstituted from DNP-16S rRNA and total 30S ribosomal proteins. DNP-30S subunits were complexed with DNP-specific IgG-antibody and examined in the electron microscope. The 3′ end of the 16S rRNA was mapped at a single region located at the inner side of the large lobe of the 30S subunit. The location of the 3′ end also provides information as to the topography of the binding domain of natural mRNA on 30S subunits, since a pyrimidine-rich sequence at the 3′ terminal region of 16S rRNA participates in the correct alignment of natural mRNAs during initiation complex formation.

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

SummaryIn 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.

[35] Localization of ribosomal proteins on the surface of ribosomal subunits from Escherichia coli using immunoelectron microscopy

Publisher Summary Immunoelectron microscopy (IEM) is one of several techniques that have proved useful for the elucidation of the structural organization of the ribosome, and has provided considerable information on the topography of the proteins on the ribosomal surface. The principle of IEM is to bind a purified IgG antibody, specific to a single ribosomal protein, to the appropriate ribosomal subunit; the bivalent antibody dimerizes two subunits which can then be examined under the electron microscope. The location of the bound antibody on the antigenic determinant of a particular protein can thus be made visible. Application of the different IEM methods described above for the localization of the ribosomal proteins on the surface of the ribosomal subunits from E. coli has led to the current model of the arrangement of the ribosomal proteins. So far, 16 proteins from the small subunit and 17 proteins from the large subunit have been localized. There is also an excellent agreement between the IEM results and the topographical data obtained by neutron scattering for the 30S subunit. If all the topographical data are taken together, the location of the remaining five proteins of the 30S subunit still to be mapped by IEM can be deduced.

A ribosomal protein that is immunologically conserved in archaebacteria, eubacteria and eukaryotes

Eubacteria Archaebacteria Eukaryote Ribosomal protein L2 Immunoblotting Evolution

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.