Reconstitution of Bacillus stearothermophilus 50 S ribosomal subunits from purified molecular components.

Bacillus stearothermophilus 50 S ribosomal subunits have been reconstituted from a mixture of purified RNA and protein components. The protein fraction of 50 S subunits was separated into 27 components by a combination of various methods including ion exchange and gel filtration chromatography. The individual proteins showed single bands in a variety of polyacrylamide gel electrophoresis systems, and nearly all showed single spots on two-dimensional polyacrylamide gels. The molecular weights of the proteins were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An equimolar mixture of the purified proteins was combined with 23 S RNA and 5 S RNA to reconstitute active 50 S subunits by the procedure of Nomura and Erdmann (Nomura, M., and Erdmann, V. A. (1970) Nature 226, 1214-1218). Reconstituted 52 S subunits containing purified proteins were slightly more active than subunits reconstituted with an unfractionated total protein extract in poly(U)-dependent polyphenylalanine synthesis and showed comparable activity in various assays for ribosomal function. The reconstitution proceeded more rapidly with the mixture of purified proteins than with the total protein extract. Reconstituted 50 S subunits containing purified proteins co-sedimented with native 50 S subunits on sucrose gradients and had a similar protein compsoition. Initial experiments on the roles of the individual proteins in ribosomal structure and function were performed. B. stearothermophilus protein 13 was extracted from 50 S subunits under the same conditions as escherichia coli L7/L12, and the extraction had a similar effect on ribosomal function. When single proteins were omitted from reconstitution mixtures, in most cases the reconstituted 50 S subunits showed decreased activity in polypheylalanine synthesis.     

Reverse phase high performance liquid chromatography of Escherichia coli ribosomal proteins: standardization of 70 S, 50 S, and 30 S protein chromatograms

We recently described the use of reverse phase high performance liquid chromatography for the separation of the proteins of the 30 S subunit of Escherichia coli ribosomes (Kerlavage, A. R., Kahan, L., and Cooperman, B. S. (1982) Anal. Biochem. 123, 342-348). In the present studies we report improvements in the technique and its extension to the separation of the proteins of the 50 S subunit and of 70 S ribosomes. Using an octadecasilyl silica column and a trifluoroacetic acid/acetonitrile solvent system, the 21 proteins of the 30 S subunit have been resolved into 17 peaks, the 33 proteins of the 50 S subunit into 22 peaks, and the 53 proteins of the 70 S ribosome into 31 peaks. The proteins present in each peak have been identified by polyacrylamide gel electrophoresis, by comparison with previously standardized chromatograms, and by calibration with authentic samples of purified proteins. All of the known ribosomal proteins have been identified on the chromatograms with the exception of L31 and its variant, L31'. Three protein peaks, not corresponding to known ribosomal proteins, have been observed in preparations from the total protein from 50 S subunits and 70 S ribosomes, but the significance of these peaks is unclear. The reverse phase high performance liquid chromatography technique has the potential for purifying all ribosomal proteins, as demonstrated by the increase in resolution we obtain when a peak isolated under standard gradient conditions and containing several proteins is reapplied to the column and eluted with a shallower gradient. Its utility in preparing proteins for functional studies is demonstrated by a reconstitution of active 30 S particles using 30 S proteins prepared by reverse phase high performance liquid chromatography.     

Purification and characterization of 50S ribosomal proteins of Escherichia coli

Summary Twenty-seven proteins of the 50S ribosomal subunit from E. coli have been purified by a combination of differential solubility in ammonium sulfate, ion-exchange chromatography, and molecular-sieve chromatography. The amino acid compositions, tryptic peptides and molecular weights of these proteins have been analyzed. Each protein is unique with respect to amino acid sequence and, according to chemical criteria, reasonably pure. The sum of the molecular weights of the twenty-seven proteins is 495000. This means that the 50S subunit could accommodate one copy of each protein.     

Rapid separation of Escherichia coli 30S ribosomal proteins by fast protein liquid chromatography (FPLC)

The proteins of the 30S ribosomal subunit from Escherichia coli have been separated by reverse-phase high-performance liquid chromatography on a short alkyl chain (C1/C8)-coated phase. The reverse-phase column was connected to a fast protein liquid chromatography (FPLC) system. The 21 proteins of the 30S ribosomal subunit were resolved into 16 peaks. Eleven proteins were isolated in purified form in a single chromatographic run as shown by polyacrylamide gel electrophoresis and amino acid analysis. Interestingly, the retention times of some proteins differed from the retention times observed on other reversed-phase support materials. The results show the speed and resolution of reverse-phase FPLC for both analytical and semi-preparative separations of 30S ribosomal proteins.     

Ionic-exchange high-performance liquid chromatography of Escherichia coli ribosomal small-subunit proteins

Ion-exchange high-performance liquid chromatography was applied to the separation of proteins from the 30S ribosomal subunit. The proteins present in each peak have been identified by polyacrylamide gel electrophoresis analysis. The purification has been made using either unmodified proteins or proteins specifically labeled at their SH group. The results clearly show that the method can be used to purify and identify ribosomal proteins.     

Ribosomes from trichomonad protozoa have prokaryotic characteristics.

1. Ribosomes from cells of the genera Trichomonas and Tritrichomonas have been isolated and characterized. The ribosomes from each organism had a sedimentation coefficient of 70S in calibrated sucrose gradients and the subunits sedimented as 50S and 30S particles under the same conditions. 2. The major ribosomal RNAs from each species were identical in size to prokaryotic ribosomal RNAs when examined by denaturing gel electrophoresis. The ribosomes contained both 5.8S and 5S RNAs. 3. The ribosomal proteins were compared by the methods of two-dimensional gel electrophoresis and reversed phase HPLC. Electrophoresis of the ribosomal proteins in two different gel systems indicated the presence of 56 proteins in T. gallinae, 40 in T. bactrachorum and 45 in the Tritrichomonas sp. The protein molecular mass range was 8.5-40 kDa. 4. The HPLC analysis confirmed the protein number established by the gel methods. 5. Both methods of analysis revealed greater similarities between the ribosomal proteins of the 2 Tritrichomonas sp. than between those of the more distantly related T. gallinae and T. bactrachorum.     

Cross-linking study on protein topography of rat liver 60 S ribosomal subunits with 2-iminothiolane

Rat liver 60 S ribosomal subunits were modified with 2-iminothiolane. After treatment with hydrogen peroxide, the cross-linked proteins were extracted and then separated into 24 fractions by chromatography on carboxymethylcellulose. Each protein fraction was then analyzed by diagonal polyacrylamide-sodium dodecyl sulfate gel electrophoresis (Sommer, A., and Traut, R.R. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 3946-3950). The pieces of gel containing cross-linked protein spots that were shifted from the diagonal line were labeled with 125I. The labeled protein was extracted from the gel and identified by three kinds of two-dimensional gel electrophoresis, followed by autoradiography. Fifty-three cross-linked protein pairs involving 35 protein species containing two acidic proteins were identified. From these and previous results, a preliminary model of the protein topography of the 60 S ribosomal subunit was constructed and discussed in relation to other functional data on 60 S ribosomal proteins.     

Ribosomal-protein synthesis is not autogenously regulated at the translational level in Xenopus laevis

Whether ribosomal-protein synthesis in Xenopus laevis is autogenously controlled at the translational level as is known to occur in prokaryotes has been studied. For this purpose ribosomal (r) proteins were prepared from X. laevis ribosomal subunits and group fractionated by ion-exchange chromatography. They were then added to an in vitro translation system directed by an oocyte mRNA fraction which contains template activity for r proteins. The synthesized radioactive products were analyzed by 2D gel electrophoresis and compared with controls. Similarly in vivo experiments were performed by microinjection of the fractionated proteins into the cytoplasm of Xenopus oocytes followed by incubation with [35S]methionine for different times. In all the experiments no evident effect of r proteins on the translation of their own mRNA was observed.     

Identification of the ribosomal proteins present in the vicinity of globin mRNA in the 40S initiation complex

The interaction of ribosomal proteins with mRNA in the 40S initiation complex was examined by chemical cross-linking. 40S initiation complexes were formed by incubating rat liver [(3)H]Met-tRNAi, rat liver 40S ribosomal subunits, rabbit globin mRNA, and partially purified initiation factors of rabbit reticulocytes in the presence of guanylyl(beta, gamma-methylene)-diphosphonate. The initiation complexes were then treated with 1,3-butadiene diepoxide to introduce crosslinks between the mRNA and proteins. The covalent mRNA-protein conjugates were isolated by chromatography on an oligo(dT) cellulose column in the presence of sodium dodecyl sulfate, followed by sucrose density gradient centrifugation. Proteins cross-linked to the mRNA were labeled with Na(125)I, extracted by extensive ribonuclease digestion, and analyzed by two-dimensional and diagonal polyacrylamide gel electrophoresis. Three ribosomal proteins, S6, S8, and S23/S24, together with small amounts of S3/S3a, S27, and S30, were identified as the protein components cross-linked to the globin mRNA protein complex, and were shown to attach directly to the mRNA. It is suggested that these proteins constitute the ribosomal binding site for mRNA in the 40S initiation complex.     

Recovery of pure ribosomal proteins from stained gels. A fast method of purification of active proteins

A simple technique has been developed for eluting ribosomal proteins from stained gels in the presence of an acetic acid solution. The ribosomal proteins are then separated from the dye by anion-exchange chromatography under dissociating conditions. Ribosomal proteins purified by these methods give total cross-reaction with proteins obtained by standard procedures, when tested by immunodiffusion against their corresponding antibodies, and show the same electrophoretic mobility as standard proteins in bidimensional polyacrylamide gel systems. Ribosomal proteins L7/L12, recovered from stained gels and purified by these methods, are able to reconstitute the elongation-factor-G-dependent GTPase activity of ribosomal particles deprived of these proteins. Radioactive protein L1, recovered in the same way, is incorporated into a total reconstituted 50-S subunit, competing with an excess of standard L1 present in the pool of total proteins from 50-S subunits used for reconstitution. These results suggest that bidimensional electrophoresis can be considered an alternative system of purification of active proteins from complex mixtures.     

A comparative study on 40S ribosomal proteins of Artemia salina and rat liver: micro analysis of amino acid composition by high-performance liquid chromatography

The amino acid compositions of 24 proteins of 40S ribosomal subunits of Artemia salina cysts were determined and compared with those of rat liver. The basic proteins of A. salina 40S ribosomes were separated by two-dimensional polyacrylamide gel electrophoresis and extracted with 70% formic acid. Samples were freed from contaminants by gel-filtration through a high-performance liquid chromatography column. Amino acid compositions were determined for individual proteins by pre-column derivatization with N,N-dimethylaminoazobenzenesulfonyl chloride followed by reverse phase high-performance liquid chromatography. The similarity of amino acid compositions between A. salina and rat liver 40S ribosomal proteins was evaluated by the method of Cornish-Bowden (Cornish-Bowden, A. (1980) Anal. Biochem. 105, 233-238), and possible relationships between A. salina and rat were detected for 16 protein species (S2, S3, S4, S6, S7, S8, S15a, S16, S17, and S18, strongly related and S14, S15, S20, S23, S24, and S26, weakly related), indicating a conservative nature of eukaryotic ribosomal proteins.     

In vivo and in vitro phosphorylation of ribosomal proteins by protein kinases from Saccharomyces cerevisiae.

From the high salt wash of the ribosomes of the yeast Saccharomyces cerevisiae, three protein kinases have been isolated and separated by DEAE-cellulose chromatography. The three kinases differ in their abilities to phosphorylate substrates such as histones (calf thymus), casein, and S. cerevisiae ribosomes; two of the kinases showed increased activity in the presence of cyclic adenosine 3',5'-monophosphate when histones and 40S ribosomal subunits were used as substrates. The protein kinases catalyzed phosphorylation of certain proteins of the 40S and 60S ribosomal subunits, and 80S ribosomes in vitro. Nine proteins of the 80S ribosome, seven proteins of the 40S subunit, and eleven of the 60S subunit were phosphorylated; different proteins were modified to various extents when different kinases were used. We have identified several proteins of 40S and 60S ribosomal subunits which are not available to the kinases in the 80S particles. Ribosomes isolated from S. cerevisiae cells growing in logarithmic phase of growth were found to contain a number of phosphorylated proteins. Studies by two-dimensional polyacrylamide gel electrophoresis indicated that the ribosomal proteins phosphorylated in vivo correspond with those phosphorylated in vitro. The relationship of in vivo phsophorylation of ribosomes to the growth and physiology of S. cerevisiae is not known.     

Identification of neighboring protein pairs in the 60 S ribosomal subunits from Saccharomyces cerevisiae by chemical cross-linking

Protein-protein cross-linking was used to determine the spatial arrangement of proteins within the 60 S ribosomal subunits of Saccharomyces cerevisiae. Protein cross-links were generated by treatment of intact ribosomal subunits with dimethyl 3,3'-dithiobispropionimidate. Proteins were extracted from the treated subunits and fractionated by Cm-cellulose chromatography. Cross-linked proteins in these fractions were analyzed by electrophoresis on two-dimensional diagonal polyacrylamide gels containing sodium dodecyl sulfate. Component members of cross-linked pairs were radiolabeled with 125I and identified by two-dimensional gel electrophoresis and comparison with nonradioactive ribosomal protein markers. Seventeen pairs involving 16 of the 45 60 S subunit proteins were identified. Several proteins were detected in numerous cross-linked dimers and were used as foci for constructing a model depicting the arrangement of proteins within the 60 S ribosomal subunit. The model also incorporated previously published data on structure and function of proteins from the yeast 60 S subunit.     

Study of the mRNA-binding region of ribosomes at different steps of translation. II. Affinity modification of Escherichia coli ribosomes by benzylidene derivative of AUGU6 in the 70S initiation complex

2',3'-O-(4-[N-(2-chloroethyl)-N-methylamino]) benzylidene derivative of AUGU6 was used for identification of the proteins in the region of the mRNA-binding centre of E. coli ribosomes. This derivative alkylated ribosomes (preferentially 30S ribosomal) with high efficiency within the 70S initiation complex. In both 30S and 50S ribosomal subunits proteins and rRNA were modified. Specificity of the alkylation of ribosomal proteins and rRNA with the reagent was proved by the inhibitory action of AUGU6. Using the method of two-dimensional electrophoresis in polyacrylamide gel the proteins S4, S12, S13, S14, S15, S18, S19 and S20/L26 which are labelled by the analog of mRNA were identified.     

A mutant from Escherichia coli which lacks ribosomal proteins S17 and L29 used to localize these two proteins on the ribosomal surface

A mutant of Escherichia coli has been isolated which lacked ribosomal proteins S17 and L29, as judged by two-dimensional gel electrophoresis. A battery of immunological tests was used to confirm this result. Ribosomes of this mutant were used as a control for the localization of proteins S17 and L29 on the surface of the ribosomal subunits of E. coli. Protein S17 has been localized on the 30S subunit body, 3-5 nm away from the lower pole, while protein L29 is located at the back of the 50S particle on the opposite side to the interface.     

Cross-linking of elongation factor 2 to rat-liver ribosomal proteins by 2-iminothiolane

Complexes containing rat liver 80S ribosomes treated with puromycin and high concentrations of KCl, elongation factor 2 (EF-2) from pig liver, and guanosine 5'-[beta, gamma-methylene]triphosphate were prepared. Neighboring proteins in the complexes were cross-linked with the bifunctional reagent 2-iminothiolane. Proteins were extracted and then separated into 22 fractions by chromatography on carboxymethylcellulose of which seven fractions were used for further analyses. Each protein fraction was subjected to diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Nine cross-linked protein pairs between EF-2 and ribosomal proteins were shifted from the line formed with monomeric proteins. The spots of ribosomal proteins cross-linked to EF-2 were cut out from the gel plate and labelled with 125I. The labelled protein was extracted from the gel and identified by three kinds of two-dimensional gel electrophoresis, followed by autoradiography. The following proteins of both large and small subunits were identified: L9, L12, L23, LA33 (acidic protein of Mr 33000), P2, S6 and S23/S24, and L3 and L4 in lower yields. The results are discussed in relation to the topographies of ribosomal proteins in large and small subunits. Furthermore we found new neighboring protein pairs in large subunits, LA33-L11 and LA33-L12.     

Isolation and identification of two protein-protein crosslinks within the two subunits of Bacillus stearothermophilus ribosomes

Bacillus stearothermophilus 30S and 50S ribosomal subunits were isolated and crosslinked with diepoxybutane. The crosslinked proteins were extracted with LiCl or with 67% acetic acid and purified by a combination of different high performance liquid chromatography techniques. The protein fractions were analysed by two-dimensional and diagonal polyacrylamide gel electrophoresis and by immunological methods. Two crosslinked protein pairs, one from the large and one from the small subunit, consisting of proteins L23-L29 and S13-S19 respectively, were isolated in milligram amounts for determination of the crosslinked amino acids.     

A comparison of ribosomal proteins from rabbit reticulocytes phosphorylated in situ and in vitro.

A comparison has been made between the ribosomal proteins phosphorylated in intact cells and proteins isolated from ribosomal subunits after modification in vitro by purified protein kinases and [gamma-32P]ATP. When intact reticulocytes were incubated for 2 h in a nutritional medium containing radioactive inorganic phosphate, one phosphorylated protein was identified as a 40S ribosomal component using two-dimensional polyacrylamide gel electrophoresis followed by electrophoresis in a third step containing sodium dodecyl sulfate. This protein, containing 99% of the total radioactivity associated with ribosomal proteins as observed by two-dimensional electrophoresis, is found in a nonphosphorylated form in addition to several phosphorylated states. These states differ by the number of phosphoryl group attached to the protein. The same 40S protein is modified in vitro by the three cAMP-regulated protein kinases from rabbit reticulocytes. Two additional proteins associated with the 40S subunit are phosphorylated in situ. These proteins migrate as a symmetrical doublet, and contain less than 1% of the radioactive phosphate in the 40S subunit. A number of phosphorylated proteins associated with 60S subunits are observed by disc gel electrophoresis after incubation of whole cells with labeled phosphate. These proteins do not migrate with previously identified ribosomal proteins and are not present in sufficient amounts to be identified as ribosomal structural proteins. Proteins in the large subunit are modified in vitro by cAMP-regulated protein kinases and ATP, and these modified proteins migrate with known ribosomal proteins. However, this phosphorylation has not been shown to occur in intact cells.     

Ribosomal proteins of HeLa cells

Ribosomal proteins from HeLa cells were analyzed by two-dimensional polyacrylamide gel electrophoresis (Kaltschmidt-Wittmann) and dodecylsulfate polyacrylamide gel electrophoresis (Laemmli). 35 proteins are associated with the small ribosomal subunit and 47 proteins with the large ribosomal subunit. The HeLa ribosomal proteins S6, S32, L40b,c, L41 and L42 are phosphorylated in vivo and in vitro. Minor differences between HeLa and rat liver ribosomal proteins were revealed by their direct coelectrophoresis.     

Mutations affecting the structural genes and the genes coding for modifying enzymes for ribosomal proteins in Escherichia coli.

Summary Temperature-sensitive mutants of an Escherichia coli K-12 strain PA3092 have been isolated following mutagenesis with nitrosoguanidine, and their ribosomal proteins analyzed by two-dimensional gel electrophoresis This method was found to be very efficient in obtaining mutants with various structural alterations in ribosomal proteins. Thus a total of some 160 mutants with alterations in 41 different ribosomal proteins have so far been isolated. By characterizing these mutants, we could isolate, not only those mutants with alterations in the structural genes for various ribosomal proteins, but also those with impairments in the modification of proteins S5, S18 and L12. Furthermore, a mutant has been obtained which apparently lacks the protein S20 (L26) with a concomitant reduction to a great extent of the polypeptide synthetic activity of the small subunit. The usefulness of these mutants in establishing the genetic architecture of the genes coding for the ribosomal proteins and their modifiers is discussed.