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G Stöffler

Publications and source records attributed to G Stöffler.

At least 73 records · Page 4Linked to original sources
PubMed1985

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.

Antibodies, Monoclonal↗
PubMed1984

Movement of the 3'-end of 16 S RNA towards S21 during activation of 30 S ribosomal subunits.

Fluorescence techniques were used to study conformational changes that occur in inactive E. coli 30 S ribosomal subunits during activation by heating in 12 mM Mg2+. Activation is associated with movement of a fluorophore on the 3'-end of 16 S RNA into a less polar environment and towards a probe on the cysteine thiol of ribosomal protein S21. The conformational change causes an apparent decrease in distance between the probes from 59 to 52 A as determined by non-radiative energy transfer.

Escherichia coli↗
PubMed1984

The NH2-terminal domain of Escherichia coli ribosomal protein L11. Its three-dimensional location and its role in the binding of release factors 1 and 2.

Ribosomes from three previously described mutants of Escherichia coli lacking L11 ( AM68 , AM76 , and AM77 ) supported in vitro termination with release factor 1 very poorly, but with release factor 2 had a severalfold elevation in activity for this function compared with ribosomes from a control strain or from a mutant containing unmethylated L11. L11 exerts its effect on the binding of the factors into a functional ribosomal complex with the termination codon. Reconstitution of L11 back into the L11-lacking ribosomes restored them to the control phenotype. The NH2-terminal part of L11 (amino acids 1-64) seems critical in modulating release factor binding. This part of L11 has been localized with the use of fragment-specific antibodies on the three-dimensional model of the 50 S subunit in the region from where the L7/L12 stalk originates. IgG antibodies from an antiserum specific for this fragment but not a middle fragment of L11 (amino acids 65-102) strongly inhibited in vitro termination. The activities of the two factors were inhibited differentially by several anti-L11 preparations recognizing antigenic determinants in the NH2-terminal part of L11. In all but one case, release factor 1 was more sensitive. These studies indicate that there are significant differences in the binding domains for the two release factors which are affected by the NH2-terminal part of L11.

Amino Acid Sequence↗
PubMed1984

The localization of protein L19 on the surface of 50 S subunits of Escherichia coli aided by the use of mutants lacking protein L19.

Three independently isolated mutants of Escherichia coli which apparently lacked protein L19 on their ribosomes, as judged by two-dimensional gels, were analyzed by a range of immunological tests to determine if the protein was indeed lacking. In two of the three, all the tests indicated that protein L19 was absent from both ribosome and supernatant. In the third, a drastically altered form of protein L19 was present on the ribosome. Electron micrographs of ribosomes obtained from the mutants were indistinguishable from those of wild type strains. The location of ribosomal protein L19 on the surface of the large subunit was determined. It was situated at the base of the 50 S particle facing the small subunit, on the side where the rod like appendage originates.

Electrophoresis, Polyacrylamide Gel↗
PubMed1984

Small-angle X-ray study of the 50 S ribosomal subunit of Escherichia coli. A comparison of different models.

A small angle X-ray scattering curve obtained from Escherichia coli 50 S subunits has been compared with the scattering curves calculated for three-dimensional 50 S models proposed by various authors. The one proposed by Lake, Stöffler and Vasiliev showed to some extent a good agreement with our experimental data. Calculating a large number of possible particle structures we found a model which optimally fits our experimental curves, but it's two-dimensional projections differ a little from the projections of 50 S subunits observed by electron microscopy.

Escherichia coli↗
PubMed1984

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

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.

Antibodies, Bacterial↗
PubMed1984

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.

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.

Antibodies, Bacterial↗
PubMed1983

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.

Antibodies↗
PubMed1983

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

Two 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.

Bacterial Proteins↗
PubMed1983

Immunological studies of Escherichia coli mutants lacking one or two ribosomal proteins.

A battery of immunological tests were used to investigate mutants which had been determined as lacking one or two ribosomal proteins on the basis of two-dimensional polyacrylamide gels. Proteins which were confirmed as missing from the ribosome in one or more mutants were large subunit proteins L1, L15, L19, L24, L27, L28, L30 and L33 and small subunit proteins S1, S9, S17 and S20. Cross-reacting material (CRM) was also absent from the post-ribosomal supernatant except in the case of protein S1. Since mutants lacking protein L11 have been previously described, any one of 13 of the 52 ribosomal proteins can be missing. None of these 13 proteins, except S1, can therefore have an indispensable role in ribosome function or assembly. In several mutants in which a protein was not missing but altered, it was present as several moieties of differing charge and size.

Bacterial Proteins↗
PubMed1983

Location of eight ribosomal proteins on the surface of the 50S subunit from Escherichia coli.

Eight ribosomal proteins, L9, L11, L15, L17, L18, L19, L23, and L29, have been localized on the surface of the 50S subunit from Escherichia coli by immunoelectron microscopy. The specificity of the antibody binding site was demonstrated by stringent absorption experiments. For each protein, the antibody attachment site was localized on the two characteristic views of the 50S subunit. Thus, each protein could be located in a confined region on the three-dimensional structural model of the 50S subunit.

Escherichia coli↗
PubMed1983

The Shine and Dalgarno hypothesis for termination: the 3' terminus of the 16S rRNA of the Escherichia coli ribosome can be modified or base-paired with a complementary oligonucleotide without affecting termination in vitro.

The occurrence of the nucleotides "...CCUUAOH" at the 3' terminus of the 16S rRNA of the small subunit of the Escherichia coli ribosome led to the suggestion that they may have a direct base pairing with the termination codon in the termination event of protein biosynthesis (Shine and Dalgarno 1974). We have examined this concept with two approaches, firstly using a 30S subunit whose 16S rRNA has been modified with a fluorescein moiety on the terminal adenosine together with the antibody against the moiety, and secondly with an oligonucleotide, UAAGG, complementary to the terminal pentanucleotide sequence of the rRNA. Collectively the data suggest that the nucleotides at the 3' terminus of 16S rRNA are not critically involved in base pairing during termination codon recognition.

Base Composition↗
PubMed1982

Ribosomal proteins cross-linked to peptide chain termination release factor 2.

The peptide chain termination factor 2 (RF2) was covalently linked to Escherichia coli ribosomal proteins with the bifunctional reagent, dimethyl suberimidate. Ribosomal RF2 complexes were identified by immunological and radioimmunological techniques which utilized both antibody specific for individual ribosomal proteins and also for RF2. Efficient cross-linking occurred with proteins L2, L7/L12, and L11 of the large ribosome subunit and to a lesser extent with S6, S17, and S18 of the small subunit. On the basis of these and other data the ribosomal binding domain of RF2 appears to be a small region at the interface between the 30 S and 50 S subunits involving parts of both subunits.

Codon↗
PubMed1981

Expression of the L11-L1 operon in mutants of Escherichia coli lacking the ribosomal proteins L1 or L11.

Using a variety of immunological techniques, the supernatant levels of ribosomal proteins were measured in mutants lacking the ribosomal proteins L1 or L11, and in wild-type strains. There was a 2.5--5-fold elevation of protein L11 level in the supernatant of strains lacking protein L1, compared to wild-type. In contrast, there was no elevation, but rather a diminution, in the corresponding L1 level in strains lacking protein L11, compared to wild-type. These results are consistent with a model for the control of expression of the L11-L1 operon in which protein L1 is an inhibitor of expression of that operon, but protein L11 is not. The supernatant concentrations of other proteins were indistinguishable in all strains.

Bacterial Proteins↗
PubMed1981

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

The 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-gamma-(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.

Escherichia coli↗