Correlation of 30S ribosomal proteins of Escherichia coli isolated in different laboratories

Summary Ribosomal proteins isolated from 30S subunits ofE. coli in four laboratories have been correlated by using two-dimensional gel electrophoresis, immunological techniques, amino acid compositions and molecular weights. The results are given in the Table. A common nomenclature for naming 30 S ribosomal proteins and their genetic loci is proposed.     

Correlation between 30S ribosomal proteins of Bacillus stearothermophilus and Escherichia coli

Summary The 30S ribosomal proteins from Bacillus stearothermophilus strains 799 and 10 were purified and correlated with those from E. coli by comparing their two-dimensional electrophoretic mobility, immunological cross-reaction, molecular weight, amino acid composition and partial amino acid sequence. A high degree of similarity was observed among the proteins from these taxonomically distant bacterial species.Paper No. 82 on Ribosomal proteins-preceding paper is by J. Horne and V. A. Erdmann, FEBS Letters, in press.N.R.C.C. No. 13514.     

Epitopes ofEscherichia coli ribosomal protein S13

To analyze the immunochemical structure ofEscherichia coli ribosomal protein S13 and its organizationin situ, we have generated and characterized 22 S13-specific monoclonal antibodies. We used a competitive enzyme-linked immunosorbent assay to divide them into groups based on their ability to inhibit binding of one another. The discovery of five groups with distinct binding properties suggested that a minimum of five distinct determinants on S13 are recognized by our monoclonal antibodies. The locations of the epitopes detected by these monoclonal antibodies have been mapped on S13 peptides. Three monoclonal antibodies bind a S13 C-terminal 34-residue segment. All the other 19 monoclonal antibodies bind a S13N-terminal segment of about 80 residues. The binding sites of these 19 monoclonal antibodies have been further mapped to subfragments of peptides. Two monoclonal antibodies recognized S13_1–22; three monoclonal antibodies bound to S13_1–40; the binding sites of three other antibodies have been located in S13_23–80, with epitopes possibly associated with residues 40–80. The remaining 11 monoclonal antibodies did not bind to these subfragments. These data provide molecular basis to the structure of S13 epitopes, whosein situ accessibility may reveal the S13 organization on the ribosome.     

Physical properties of ribosomal proteins isolated under different conditions from the Escherichia coli 50 S subunit

Physical properties of ribosomal proteins obtained with or without denaturating agents were compared. CD measurements and NMR studies have shown that proteins L2, L19, L24 and L30 isolated under denaturing conditions have the same properties as those prepared avoiding denaturating agents. CD and NMR spectra of proteins L1, L6, L11, L23, L25 and L29 obtained by us under denaturating conditions practically coincide with the data for the same proteins reported under 'mild' conditions. These findings suggest that the differences of reported physical properties can be due to different procedures of protein renaturation rather than to the methods of their isolation.     

Immunoprecipitation of 70S, 50S and 30S ribosomes ofEscherichia coli

Antibodies were raised in rabbits against 70S ribosomes, 50S and 30S ribosomal subunits individually. Purified immunoglobulins from the antiserum against each of the above ribosomal entities were tested for their capabilities of precipitating 70S, 50S and 30S ribosomes. The observations revealed the following: (i) The antiserum (IgG) raised against 70S ribosomes precipitates 70S ribosomes completely, while partial precipitation is seen with the subunits, the extent of precipitation being more with the 50S subunits than with 30S subunits; addition of 50S subunits to the 30S subunits facilitates the precipitation of 30S subunits by the antibody against 70S ribosomes. (ii) Antiserum against 50S subunits has the ability to immunoprecipitate both 50S and 70S ribosomes to an equal extent. (iii) Antiserum against 30S subunits also has the property of precipitating both 30S and 70S ribosomes. The differences in the structural organisation of the two subunits may account for the differences in their immunoprecipitability.     

Purification of Escherichia coli 30S ribosomal proteins by high performance liquid chromatography

High performance liquid chromatography was applied to the separation of proteins derived from the Escherichia coli 30S ribosomal subunit. Several methods of separating this protein mixture has been tested: size-exclusion chromatography on hydrophilic phases; ion exchange and reversed phase chromatography (on C2 to C18 hydrocarbon-bonded supports). Various elution systems were examined in order to obtain pure proteins suitable for micro-sequence analysis. The resolution and yields of the proteins varied considerably, depending on the type of support and gradient system used. The best results were achieved with uniformly globular-shaped supports of large pore size, and by combining high performance size exclusion with rechromatography on reversed phase columns. Purification conditions for the individual proteins are listed. The methods employed avoid any precipitation step and allow easy identification of the proteins by one or two-dimensional gel electrophoresis, amino-acid analysis or direct manual or automatic micro-sequencing. Since the isolation time is much reduced compared with conventional purification procedures, the proteins obtained by the techniques described here are well suited for topographical and immunological studies or reconstitution assays. Ribosomal proteins of other organisms can be separated under similar conditions.     

Fast preparative separation of 'native' core E coli 30S ribosomal proteins.

We have developed an ion-exchange high performance liquid chromatographic method for preparative separation of 'core' proteins from E coli 30S ribosomal subunits, extracted with salt under non-denaturing conditions. This method yields individual proteins in pure and native form at high concentrations, (5 to 25 mg/ml) suitable for direct use in 1D-, 2D- or 3D-NMR studies.     

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.