Mapping proteins of the 50S subunit from Escherichia coli ribosomes

Mapping of protein positions in the ribosomal subunits was first achieved for the 30S subunit by means of neutron scattering about 15 years ago. Since the 50S subunit is almost twice as large as the 30S subunit and consists of more proteins, it was difficult to apply classical contrast variation techniques for the localisation of the proteins. Polarisation dependent neutron scattering (spin-contrast variation) helped to overcome this restriction. Here a map of 14 proteins within the 50S subunit from Escherichia coli ribosomes is presented including the proteins L17 and L20 that are not present in archeal ribosomes. The results are compared with the recent crystallographic map of the 50S subunit from the archea Haloarcula marismortui.     

Identification of the 30 S protein adjacent to peptidyl transferase catalytic center of Escherichia coli ribosomes

Iodoacetylphenylalanyl-tRNA^Phe was used as an affinity label to localize the ribosomal components involved in the peptidyl transferase catalytic center of Escherichia coli ribosomes. When labeling was carried out at pH 5.0, the affinity label could specifically label the ribosomal components which comprise the catalytic center. Analysis of ribosomal proteins which had reacted with the affinity label revealed that a 30 S subunit protein, S 20, was located at or near to the ribosomal binding site of the 3-terminus of aminoacyl- or peptidyl-tRNA.     

Monoclonal antibodies to Escherichia coli 50S ribosomes.

Hybridoma cell lines that produce monoclonal antibodies directed against 50S Ribosomal proteins have been isolated. Spleen cells (from BALB/c mice immunized with 50S ribosomal subunits extracted from Escherichia coli) were fused to mouse myeloma cell line SP2/O-Ag 14. The initial screening for antibody producing hybridomas was carried out by a double antibody sandwich method; hybridomas were subsequently cloned in soft agar. Antibodies were characterized by their specific binding to individual 50S ribsomal proteins separated on phosphocellulose columns and in two-dimensional polyacrylamide gels. The assignments were confirmed with purified single ribosomal proteins. Of four clones analyzed thus far, two are identical with specificity for r-protein L5. The other clones produce two different antibodies directed against r-protein L20. Each monoclonal antibody formed ribosome dimers visualizable in the electron microscope. Dimers could be reacted with a different second antibody to form chains containing 8 or more ribosomes, which may be useful for structural studies.     

Structure of the Escherichia coli 50 S ribosomal subunit

Freeze-dried and shadowed Escherichia coli 50 S ribosomal subunits have been examined by electron microscopy and a model of the subunit has been constructed. High resolution shadow casting has enabled us to determine independently the absolute hand of the subunit and to reveal some new structural features.     

Copies of protein S6 in Escherichia coli ribosomes

A reinvestigation of the copy number of protein S6 in Escherichia coli ribosomes shows that there is only one copy of S6 in 30 S subunits and in 70 S ribosomes. The previously reported higher value (Subramanian, A. R. (1980) J. Biol. Chem. 255, 6941-6946) is shown to arise from the presence in the usual ribosome preparations of a protein which co-electrophoreses with S6 but does not react with S6 antiserum. This protein is removed when ribosomes are purified by passage through a sucrose gradient.     

Single protein omission reconstitution studies of tetracycline binding to the 30S subunit of Escherichia coli ribosomes

In previous work we showed that on photolysis of Escherichia coli ribosomes in the presence of [3H]tetracycline (TC) the major protein labeled is S7, and we presented strong evidence that such labeling takes place from a high-affinity site related to the inhibitory action of TC [Goldman, R. A., Hasan, T., Hall, C. C., Strycharz, W. A., & Cooperman, B. S. (1983) Biochemistry 22, 359-368]. In this work we use single protein omission reconstitution (SPORE) experiments to identify those proteins that are important for high-affinity TC binding to the 30S subunit, as measured by both cosedimentation and filter binding assays. With respect to both sedimentation coefficients and relative Phe-tRNAPhe binding, the properties of the SPORE particles we obtain parallel very closely those measured earlier [Nomura, M., Mizushima, S., Ozaki, M., Traub, P., & Lowry, C. V. (1969) Cold Spring Harbor Symp. Quant. Biol. 34, 49-61], with the exception of the SPORE particle lacking S13. A total of five proteins, S3, S7, S8, S14, and S19, are shown to be important for TC binding, with the largest effects seen on omission of proteins S7 and S14. Determination of the protein compositions of the corresponding SPORE particles demonstrates that the observed effects are, for the most part, directly attributable to the omission of the given protein rather than reflecting an indirect effect of omitting one protein on the uptake of another. A large body of evidence supports the notion that four of these proteins, S3, S7, S14, and S19, are included, along with 16S rRNA bases 920-1396, in one of the major domains of the 30S subunit.     

50-S subunit from Escherichia coli ribosomes. Isolation of active ribosomal proteins and protein complexes.

A method is described for the isolation of highly purified proteins from the 50-S subunit of Escherichia coli ribosomes. All the proteins from the large subunit could be isolated with the exception of L14, L26, L31 and L34. The isolated proteins are functionally active in reconstituted particles. The method consists of successive NH4Cl/EtOH and LiCl washing steps, which split off distinct groups of proteins from the ribosome. The protein groups are further separated by a combination of gel filtration (Sephadex G-100) and ion-exchange chromatography (carboxymethylcellulose) in the presence of 6 M urea, at neutral pH and 4 degrees C. The purity of the proteins was analyzed by two-dimensional gel electrophoresis. In addition, ten protein complexes were isolated and identified.     

Shape of protein L11 from the 50S ribosomal subunit of Escherichia coli.

Protein L11 from the 50S ribosomal subunit of Escherichia coli A19 was purified by a method using nondenaturing conditions. Its shape in solution was studied by hydrodynamic and low-angle x-ray scattering experiments. The results from both methods are in good agreement. In buffers similar to the ribosomal reconstitution buffer, the protein is monomeric at concentrations up to 3 mg/mL and has a molecular weight of 16 000-17 000. The protein molecule resembles a prolate ellipsoid with an axial ratio of 5-6:1 a radius of gyration of 34 A, and a maximal length of 150 A. From the low-angle x-ray diffraction data, a more refined model of the protein molecule has been constructed consisting of two ellipsoids joined by their long axes.     

Modification of histidine residues on proteins from the 50S subunit of the Escherichia coli ribosome. Effects on subunit assembly and peptidyl transferase centre activity.

L2, L3, L4, L16 and L20 are proteins of the 50S ribosomal subunit of Escherichia coli which are essential for the assembly and activity of the peptidyl transferase centre. These proteins have been modified with the histidine-specific reagent, diethylpyrocarbonate, while L17 and L18 were treated as controls. Each modified protein tested was able to participate in the reconstitution of a 50S particle when replacing its normal counterpart, although the particles assembled with modified L2 were heterogeneous. However, although they could support assembly, modified L16 and L20 were not themselves reconstituted stably, and modified L2 and L3 were found in less than stoichiometric amounts. Particles assembled in the presence of modified L16 retained significant peptidyl transferase activity (60-70% at 10 mM diethylpyrocarbonate) whereas those reconstituted with modified L2, L3, L4 or L20 had low activity (10-30% at 10 mM diethylpyrocarbonate). The particles assembled with the modified control protein, L17, retained 80% of their peptidyl transferase activity under the same conditions. The histidine residues within the essential proteins therefore contribute to ribosome structure and function in three significant ways; in the correct assembly of the ribosomal subunit (L2), for the stable assembly of the proteins within the ribosomal particle (L20 and L16 in particular), and directly or indirectly for the subsequent activity of the peptidyl transferase centre (L2, L3, L4 and L20). The essential nature of the unmodified histidines for assembly events precludes the use of the chemical-modification strategy to test the proposal that a histidine on one of the proteins might participate in the catalytic activity of the centre.     

Kinetic properties of Escherichia coli ribosomes with altered forms of S12.

E. coli ribosomes with alterations in S12 leading to streptomycin resistance (SmR), dependence (SmD) and pseudodependence (SmP) were studied with the quench-flow technique. Kinetic changes at the various steps of the elongation cycle were identified. The rate of hydrolysis of GTP in the ternary complex in the ribosomal A-site is decreased drastically in SmD and moderately in SmP in relation to wild-type ribosomes. Addition of streptomycin restores much of the wild-type behaviour. The SmD, SmP and SmR ribosomes have an enhanced GTP-hydrolysis idling reaction on EF-Tu, which is correlated with how aggressive proofreaders these ribosomes are in steady-state assays. We use our in vitro findings to discuss the in vivo physiology of these mutants as well as mechanistic features of E. coli translation.     

Ribosomal assembly deficiency in an Escherichia coli thermosensitive mutant having an altered L24 ribosomal protein.

Localized P1 mutagenesis was used to screen for conditionally lethal mutations in ribosomal protein genes. One such mutation, 2859mis, has been mapped inside the ribosomal protein gene cluster at 72 minutes on the Escherichia coli chromosome and cotransduces at 98% with rpsE (S5). The 2869mis mutation leads to thermosensitivity and impaired assembly in vivo of 50 S ribosomal particles at 42 °C. The strain carrying the mutation has an altered L24 ribosomal protein which at 42 °C shows weaker affinity for 23 S RNA than the wild-type protein. The mutational alteration involves a replacement of glycine by aspartic acid in protein L24 from the mutant. We conclude therefore that the 2859mis mutation affects the structural gene for protein L24 (rplX).     

The composition of an unusual precursor of 50 S ribosomes in a mutant of Escherichia coli.

Escherichia coli strain 15--28 is a mutant which during exponential growth contains large amounts of a '47S' ribonucleoprotein precursor to 50S ribosomes. The '47S particles' are more sensitive to ribonuclease than are 50S ribosomes. The 23 S RNA of 47S particles may be slightly undermethylated, but cannot be distinguished from the 23S RNA of 50S ribosomes by sedimentation or electrophoresis. Isolated particles have 10--15% less protein than do 50S ribosomes; proteins L16, L28 and L33 are absent. Comparison with precursor particles studied by other workers in wild-type strains of E. coli suggests that the assembly of 50S ribosomes in strain 15--28 is atypical.     

Identification of 50S components neighboring 23S rRNA nucleotides A2448 and U2604 within the peptidyl transferase center of Escherichia coli ribosomes

The 23S rRNA nucleotides 2604-12 and 2448-58 fall within the central loop of domain V, which forms a major part of the peptidyl transferase center of the ribosome. We report the synthesis of radioactive, photolabile 2'-O-methyloligoRNAs, PHONTs 1 and 2, complementary to these nucleotides and their exploitation in identifying 50S ribosomal subunit components neighboring their target sites. Photolysis of the 50S complex with PHONT 1, complementary to nts 2604-12, leads to target site-specific photoincorporation into protein L2 and 23S rRNA nucleotides A886, Alpha1918, A1919, G1922-C1924, U2563, U2586, and C2601. Photolysis of the 50S complex with PHONT 2, complementary to nts 2448-58, leads to target site-specific probe photoincorporation into proteins L2, L3, one or more of proteins L17, L18, L21, and of proteins L9, L15, L16, and 23S rRNA nucleotides C2456 and psi2457. Chemical footprinting studies show that 2'-O-methyloligoRNA binding causes little distortion of the peptidyl transferase center but do provide suggestive evidence for the location of flexible regions within 23S rRNA. The significance of these results for the structure of the peptidyl transferase center is considered.     

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.     

The structural gene for the ribosomal protein S18 in Escherichia coli. II. Chemical studies on the protein S18 having an altered electrophoretic mobility

A mutant of Escherichia coli strain CR341 has an altered 30 S ribosomal protein S18. The alteration involves a change in the electrophoretic mobility of S18. S18 proteins were purified from the mutant and the parent strain, respectively, and their amino acid composition and tryptic peptides were compared. The results have shown that the mutational alteration involves substitution of cysteine for arginine. In addition, we determined the electrophoretic mobility of S18 proteins modified by ethyleneimine. The modification, which involves conversion of cysteine residues to S-(2-aminoethyl)cysteine, causes a greater electrophoretic mobility increase in the mutant protein than in the wild type protein, resulting in identical mobilities for the aminoethylated proteins. This experiment gives further support to the conclusion that the original mobility difference between mutant and wild type proteins is due to the mutational substitution of cysteine for arginine. The S18 obtained from a recombinant was also studied. The recombinant protein was found to have the mobility of the wild type protein and the wild type primary structure, as judged by amino acid composition and tryptic peptide analysis. This recombinant was obtained from the mutant by introducing Hfr strain G10 chromosome segments in the region between 70 and 10 minutes, and not in the str-spc region at 64 minutes, as described in the preceding paper. These results, together with those in the preceding paper, show that the mutation studied here is in the structural gene for S18, and that it maps outside the str-spc region.