Purification of ribosomal proteins from Escherichia coli by cation exchange and reversed phase FPLC

A complex mixture of 21 proteins from the 30S ribosomal subunit of Escherichia coli was fractionated on a cation-exchanger, then further separated on a C8 reversed-phase column. A set of 14 proteins were purified to homogeneity. The same protein mixture was also analysed on a C8 RPC column using a triethylamine phosphate (TEAP, pH2.2)/acetonitrile or a trifluoroacetic acid/acetonitrile solvent system which gave 11 and 8 purified proteins, respectively. Altogether, 16 out of 21 proteins from the 30S ribosomal subunit were purified.     

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.     

Identification of the ribosomal proteins phosphorylated by the ribosome-associated casein kinase type II from cryptobiotic gastrulae of the brine shrimp Artemia sp

Phosphorylation of the ribosomal proteins by the extra-ribosomal protein kinase was investigated "in situ" and with purified 40 S or 60 S ribosomal proteins from cryptobiotic embryos of Artemia sp. Ribosomal proteins that were most readily phosphorylated in 80 S ribosomes included S6 and S8 of the 40 S subunit and proteins L9, L13 and L18 of the 60 S subunit. Several additional polypeptides were phosphorylated when purified 40 S or 60 S ribosomal proteins were separately incubated in the reconstituted system. The possible functions of ribosomal phosphorylation in protein synthesis will be discussed.     

Crosslinking of eukaryotic initiation factor eIF3 to the 40S ribosomal subunit from rabbit reticulocytes

Complexes of purified 40S ribosomal subunits and initiation factor 3 from rabbit reticulocytes were crosslinked using the reversible protein crosslinking reagent, 2-iminothiolane, under conditions shown previously to lead to the formation of dimers between 40S proteins but not higher multimers. The activity of both the 40S subunits and initiation factor 3 was maintained. Protein crosslinked to the factor was purified by sucrose density gradient centrifugation following nuclease digestion of the ribosomal subunit: alternatively, the total protein was extracted from 40S: factor complexes. The protein obtained by either method was analyzed by two-dimensional diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Ribosomal proteins were found in multimeric complexes of high molecular weight due to their crosslinking to components of eIF3. Identification of the ribosomal proteins appearing below the diagonal was accomplished by elution, radioiodination, two-dimensional polyacrylamide/urea gel electrophoresis, and radioautography. Proteins S2, S3, S3a, S4, S5, S6, S8, S9, S11, S12, S14, S15, S16, S19, S24, S25, and S26 were identified. Because many of the proteins in this group form crosslinked dimers with each other, it was impossible to distinguish proteins directly crosslinked to eIF3 from those crosslinked indirectly through one bridging protein. The results nonetheless imply that the 40S ribosomal proteins identified are at or near the binding site for initiation factor 3.     

Influence of the state of ribosome association on the phosphorylation of ribosomal proteins in isolated ribosome--protein kinase systems from rat cerebral cortex.

Ribosomal protein phosphorylation was investigated in isolated ribosomal subunits and polyribosomes from rat cerebral cortex in the presence of [gamma-32P]ATP and purified catalytic subunit of cyclic AMP-dependent protein kinase from the same tissue. Ribosomal proteins that were most readily phosphorylated in isolated cerebral ribosomal subunits included proteins S2, S3a, S6 and S10 of the 40 S subunit and proteins L6, L13, L14, L19 and L29 of the 60 S subunit. These proteins were also phosphorylated in cellular preparations of rat cerebral cortex in situ or in vitro [Roberts & Ashby (1978) J. Biol. Chem. 253, 288-296; Roberts & Morelos (1979) Biochem. J. 184, 233-244]. However, several additional ribosomal proteins were phosphorylated when isolated 40 S or 60 S subunits were separately incubated in the reconstituted system. Analogous results were obtained with an equimolar mixture of cerebral 40 S and 60 S subunits under comparable conditions. In contrast, extensive exposure of purified cerebral polyribosomes to the catalytic subunit resulted in phosphorylation of only those ribosomal proteins of the 40 S subunit that were most highly labelled after the administration of [32P]Pi in vivo: proteins S2, S6 and S10. Ribosomal proteins of 60 S subunits that were readily phosphorylated in isolated cerebral polyribosomes included proteins L6, L13 and L29. These results indicate that polyribosome formation markedly decreases the number of ribosomal protein sites available for phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase. Moreover, the findings suggest that, of the ribosomal protein phosphorylations observed in rat cerebral cortex in vivo, proteins S2, S6, S10, L6, L13 and L29 can be phosphorylated in polyribosomes, whereas proteins S3a, S5, L14 and L19 may become phosphorylated only in free ribosomal subunits.     

Efficient reconstitution of functional Escherichia coli 30S ribosomal subunits from a complete set of recombinant small subunit ribosomal proteins.

Previous studies have shown that the 30S ribosomal subunit of Escherichia coli can be reconstituted in vitro from individually purified ribosomal proteins and 16S ribosomal RNA, which were isolated from natural 30S subunits. We have developed a 30S subunit reconstitution system that uses only recombinant ribosomal protein components. The genes encoding E. coli ribosomal proteins S2-S21 were cloned, and all twenty of the individual proteins were overexpressed and purified. Reconstitution, following standard procedures, using the complete set of recombinant proteins and purified 16S ribosomal RNA is highly inefficient. Efficient reconstitution of 30S subunits using these components requires sequential addition of proteins, following either the 30S subunit assembly map (Mizushima & Nomura, 1970, Nature 226:1214-1218; Held et al., 1974, J Biol Chem 249:3103-3111) or following the order of protein assembly predicted from in vitro assembly kinetics (Powers et al., 1993, J MoI Biol 232:362-374). In the first procedure, the proteins were divided into three groups, Group I (S4, S7, S8, S15, S17, and S20), Group II (S5, S6, S9, Sll, S12, S13, S16, S18, and S19), and Group III (S2, S3, S10, S14, and S21), which were sequentially added to 16S rRNA with a 20 min incubation at 42 degrees C following the addition of each group. In the second procedure, the proteins were divided into Group I (S4, S6, S11, S15, S16, S17, S18, and S20), Group II (S7, S8, S9, S13, and S19), Group II' (S5 and S12) and Group III (S2, S3, S10, S14, and S21). Similarly efficient reconstitution is observed whether the proteins are grouped according to the assembly map or according to the results of in vitro 30S subunit assembly kinetics. Although reconstitution of 30S subunits using the recombinant proteins is slightly less efficient than reconstitution using a mixture of total proteins isolated from 30S subunits, it is much more efficient than reconstitution using proteins that were individually isolated from ribosomes. Particles reconstituted from the recombinant proteins sediment at 30S in sucrose gradients, bind tRNA in a template-dependent manner, and associate with 50S subunits to form 70S ribosomes that are active in poly(U)-directed polyphenylalanine synthesis. Both the protein composition and the dimethyl sulfate modification pattern of 16S ribosomal RNA are similar for 30S subunits reconstituted with either recombinant proteins or proteins isolated as a mixture from ribosomal subunits as well as for natural 30S subunits.     

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.     

Homology between Drosophila melanogaster and Escherichia coli ribosomal proteins

Summary Antibodies raised against D. melanogaster ribosomal proteins were used to examine possible structural relationships between eukaryotic and prokaryotic ribosomal proteins. The antisera were raised against either groups of ribosomal proteins or purified individual ribosomal proteins from D. melanogaster. The specificity of each antiserum was confirmed and the identity of the homologous E. coli ribosomal protein was determined by immunochemical methods. Immuno-overlay assays indicated that the antiserum against the D. melanogaster small subunit protein S14 (anti-S14) was highly specific for protein S14. In addition, anti-S14 showed a cross-reaction with total E. coli ribosomal proteins in Ouchterlony double immunodiffusion assays and with only E. coli protein S6 in immuno-overlay assays. From these and other experiments with adsorption of anti-S14 with individual purified proteins, the E. coli protein homologous to the D. melanogaster protein S14 was established as protein S6.     

Binding of 16S rRNA to chloroplast 30S ribosomal proteins blotted on nitrocellulose

Protein-RNA associations were studied by a method using proteins blotted on a nitrocellulose sheet. This method was assayed with Escherichia Coli 30S ribosomal components. In stringent conditions (300 mM NaCl or 20° C) only 9 E. coli ribosomal proteins strongly bound to the 16S rRNA: S4, S5, S7, S9, S12, S13, S14, S19, S20. 8 of these proteins have been previously found to bind independently to the 16S rRNA. The same method was applied to determine protein-RNA interactions in spinach chloroplast 30S ribosomal subunits. A set of only 7 proteins was bound to chloroplast rRNA in stringent conditions: chloroplast S6, S10, S11, S14, S15, S17 and S22. They also bound to E. coli 16S rRNA. This set includes 4 chloroplast-synthesized proteins: S6, S11, S15 and S22. The core particles obtained after treatment by LiCl of chloroplast 30S ribosomal subunit contained 3 proteins (S6, S10 and S14) which are included in the set of 7 binding proteins. This set of proteins probably play a part in the early steps of the assembly of the chloroplast 30S ribosomal subunit.     

Ordered multisite phosphorylation of Xenopus ribosomal protein S6 by S6 kinase II.

Phosphorylated ribosomal proteins were isolated from Xenopus 40 S ribosomal subunits by reversed-phase high performance liquid chromatography (HPLC) to enable direct analysis of the phosphorylation sites in ribosomal protein S6. Xenopus S6 closely resembled mammalian S6 with respect to the following properties: (i) reversed-phase HPLC elution behavior, (ii) amino-terminal sequence (96% identity in the first 37 residues), and (iii) an identical sequence within the region of its phosphorylation sites. Whereas S6 was the only ribosomal protein phosphorylated in vitro by Xenopus S6 kinase II, ribosomes phosphorylated in vivo were found to be associated with an additional phosphoprotein having an amino-terminal sequence identical to that of the ubiquitin carboxyl-terminal extension protein CEP 80. S6 kinase II phosphorylated at least four sites (serines 1-3 and 5) in the sequence Arg-Arg-Leu-Ser(1)-Ser(2)-Leu-Arg-Ala-Ser(3)-Thr-Ser(4)-Lys-Ser(5)-, which correspond to the residues known to be phosphorylated in the carboxyl-terminal region of mammalian S6. The in vivo S6 phosphorylation sites in maturing Xenopus oocytes were shown to be located within the same cluster of serine residues, although individual sites were not identified. Kinetic analysis of S6 kinase II-catalyzed phosphorylation events indicated a simple sequential mechanism of multisite phosphorylation initiating at either serine 2 (preferred) or serine 1, with the rates of phosphorylation of individual sites occurring in the order serine 2 greater than serine 1 greater than serine 3 greater than serine 5.     

Cerebral ribosomal protein phosphorylation in experimental hyperphenylalaninaemia.

Investigations were carried out on the effects of phenylalanine loading on ribosomal protein phosphorylation in cerebral cortices of infant rats. Administration of L-phenylalanine intraperitoneally, in doses of 1 or 2 mg/g body wt., resulted within 30 min in a significant decrease in incorporation of radioactivity from intracisternally administered [32P]Pi into constitutive ribosomal proteins of the cerebral 40S subunit. This phenomenon was not accompanied by significant variations in 32P uptake into the cerebral cytosol. Incorporation of radioactivity into ribosomal proteins of the cerebral 60S subunit exhibited only minor variations under these circumstances. Alterations in the phosphorylation state of cerebral 40S ribosomal proteins induced by phenylalanine loading involved principally the S6 protein, which exists in multiple states of phosphorylation. The proportions of the more highly phosphorylated congeners of this protein were markedly decreased, as detected by two-dimensional electrophoretograms and autoradiographs of the cerebral 40S ribosomal proteins. Phenylalanine loading also altered the relative extent of phosphorylation of the S6 protein in cerebral polyribosomes and monoribosomes. In control animals, the specific radioactivity of 40S proteins in cerebral polyribosomes was five to ten times that of 40S proteins in the monoribosome population. At 1 h after phenylalanine administration, the specific radioactivities of 40S proteins in the two ribosome populations tended to approach equality. These alterations in ribosomal protein phosphorylation were accompanied by a decrease in the proportion of polyribosomes in purified ribosome preparations isolated from cerebral cortices of phenylalanine-treated infant rats. In animals given the higher dose of phenylalanine (2 mg/g body wt.), subsequent administration of a mixture of seven neutral amino acids, which resulted in partial recovery of polyribosomes, also tended to reverse the changes in ribosomal protein phosphorylation. Variations in the activities of ribonuclease enzymes in the cerebral cytosol were also observed under these conditions. Administration of phenylalanine increased the activities of cerebral ribonucleases, whereas subsequent treatment with the amino acid mixture partly reversed this effect. The results suggest that alterations in cerebral ribosomal protein phosphorylation, ribosome aggregation and ribosome function are interrelated in experimental hyperphenylalaninaemia.     

Purification and properties of a ribosomal protein methylase from Eschericha coli Q13.

Ribosomal protein methylase has been purified from Escherichia coli strain Q13 using methyl-deficient 50S subunits as substrates. The purified enzyme (or enzyme complex) which is devoid of rRNA methylating activity is quite stable and has a pH optimum around 8.0. The Km for S-adenosyl-L-methionine is 3.2 muM. The molecular weight of the enzyme is 3.1 X 10(4); minor methylating activity was also detected for protein peaks with molecular weights of 1.7 X 10(4) and 5.6 X 10(4). Protein L11 is the major protein methylated by the purified enzyme. Product analysis revealed the presence of N epislon-trimethyllysine, a methylated neutral amino acid(s) previously observed in protein L11 and N epislon-monomethyllysine. Free ribosomal proteins were much better substrates for the methylation, indicating that methylation of 50S ribosomal proteins can occur before the complete assembly of the 50S ribosomal subunit.     

Individual ribosomal protein pool size and turnover rate in Escherichia coli

The pool size of free individual ribosomal proteins present in the cell sap of Escherichia coli has been determined by pulse-labelling a culture before a chase with cold marker.Ribosomes plus ribosomal precursor particles were prepared together and the proteins from this fraction purified. The specific radioactivity of each 30 S and 50 S protein was measured at the time of pulse and at the various times of chase: unequal labelling was already observed at the time of pulse; the kinetics of chase of most 30 S proteins reached a plateau very rapidly; the kinetics of 50 S proteins were more variable. Precise calculation of individual pool size was carried out using the mathematical model described in the Appendix. Almost all ribosomal 30 S proteins have a pool size close to zero. Only four 30 S proteins (S10, S16, S17 and S18) have a sizeable pool (2 to 6% of the corresponding ribosomal protein). Most 50 S proteins have a small pool size (1 to 2%). The free ribosomal proteins of the pool are transferred to mature ribosomes; the half-life of these proteins in the pool has been calculated (0 to 1·4 min). Finally, as judged from the kinetic data, no degradation of ribosome-bound protein was apparent. The significance of the results is discussed with respect to the function of ribosome and the process of ribosome biogenesis.     

The amino-acid sequences of the Bacillus stearothermophilus ribosomal proteins S17 and S21 and their comparison to homologous proteins of other ribosomes.

Ribosomal proteins S17 and S21 from the moderate thermophile Bacillus stearothermophilus were purified by one-step high-performance liquid chromatography from the 30S-subunit protein mixture employing a semi-preparative reversed-phase C4 column. The complete amino-acid sequences of these proteins were determined by a combination of N-terminal sequencing in picomole quantities of the protein and of appropriate peptide fragments. Proteins S17 and S21 consist of 86 and 55 amino-acid residues, corresponding to molecular masses of 10074 and 6593 Da, respectively. They are homologous to proteins S17 and S21 from the Escherichia coli ribosome, showing 50 and 55% identities in the corresponding regions, respectively. The C-terminal region of protein S21 from B. stearothermophilus has a deletion of 15 residues as compared to the E. coli S21 protein. The evolutionary relationships of the Bacillus proteins to various other members of the S17 and S21 ribosomal protein families are discussed.     

Isolation of a cloned DNA segment containing a ribosomal protein gene of Drosophila melanogaster

A Drosophila genomic DNA library in the vector Charon 4 was screened using cDNA derived from the small (6S-12S) poly(A)+ mRNA of 2-6-h-old Drosophila embryos. This fraction of mRNA is enriched for ribosomal protein-coding sequences. The selected recombinants were hybridized to total mRNA under conditions which allowed for isolation of homologous mRNAs. The mRNA from these RNA/DNA hybrids was eluted and translated in vitro. The translation products were analyzed by one- and two-dimensional electrophoresis with authentic ribosomal proteins as standards. One cloned DNA segment was found to contain a ribosomal protein gene, and a sequence which hybridizes strongly to at least 5 other ribosomal protein mRNAs.     

Binding of rat ribosomal proteins to yeast 5.8S ribosomal ribonucleic acid.

5.8 S RNA-protein complexes were prepared using purified yeast 5.8 S RNA and proteins from the large ribosomal subunit of rat liver. Formation of such hybrid complexes, as measured by Millipore filtration, was dependent on protein concentration. Binding of proteins to the RNA could approach saturation. Such complexes were isolated from sucrose density gradient centrifugation and shown to contain proteins L6, L8, L19, L35 and L35a. These proteins were identified by their molecular weights on polyacrylamide gels containing dodecylsulfate and their mobilities on two dimensional polyacrylamide gels.     

Identification of fifteen neighboring protein pairs in the Escherichia coli 50 S ribosomal subunit crosslinked with 2-iminothiolane.

The 50 S ribosomal subunit of Escherichia coli was allowed to react with 2-iminothiolane under conditions in which amidine-linked sulfhydryl derivatives were formed between lysine ?-amino groups in ribosomal proteins and the heterocyclic thioimidate. Crosslinking between sulfhydryl groups close enough to form intermolecular disulfide bonds was promoted by oxidation of the modified ribosomal subunits. Disulfide-linked dimers were partially purified by extraction of the oxidized subunits with lithium chloride and electrophoresis of the salt-extracted fractions in polyacrylamide/urea gels at pH 5.5. Crosslinked protein dimers were separated by polyacrylamide/sodium dodecyl sulfate diagonal gel electrophoresis. Fifteen protein dimers were identified. Many of them involve proteins implicated in functional sites of the 50 S subunit and in ribosome assembly. The crosslinking results show the proximity of many of these proteins at these active centers, and extend the neighborhood by demonstrating the presence of additional proteins.     

Preparative ion-exchange high-performance liquid chromatography of bacterial ribosomal proteins

We have developed analytical and preparative ion-exchange HPLC methods for the separation of bacterial ribosomal proteins. Proteins separated by the TSK SP-5-PW column were identified with reverse-phase HPLC and gel electrophoresis. The 21 proteins of the small ribosomal subunit were resolved into 18 peaks, and the 32 large ribosomal subunit proteins produced 25 distinct peaks. All peaks containing more than one protein were resolved using reverse-phase HPLC. Peak volumes were typically a few milliliters. Separation times were 90 min for analytical and 5 h for preparative columns. Preparative-scale sample loads ranged from 100 to 400 mg. Overall recovery efficiency for 30S and 50S subunit proteins was approximately 100%. 30S ribosomal subunit proteins purified by this method were shown to be fully capable of participating in vitro reassembly to form intact, active ribosomal subunits.     

Comparative study between prokaryotes and eukaryotes by chemical iodination of ribosomal proteins.

Escherichia coli and Saccharomyces cerevisiae ribosomal proteins were chemically iodinated with 125I by chloramine T under conditions in which the proteins were denatured. The labelled proteins were subsequently separated by two-dimensional gel electrophoresis with an excess of untreated ribosomal proteins from the same species. The iodination did not change the electrophoretic mobility of the proteins as shown by the pattern of spots in the stained gel slabs and their autoradiography. The 125I radioactivity incorporated in the proteins was estimated by cutting out the gel spots from the two-dimensional electrophoresis gel slabs. The highest content of 125I was found in the ribosomal proteins L2, L11, L13, L20/S12, S4 and S9 from E. coli, and L2/L3, L4/L6/S7, L5, L19/L20, L22/S17, L29/S27, L35/L37 and S14/S15 from S. cerevisiae. Comparisons between the electrophoretic patterns of E. coli and S. cerevisiae ribosomal proteins were carried out by coelectrophoresis of labelled and unlabelled proteins from both species. E. coli ribosomal proteins L5, L11, L20, S2, S3 and S15/S16 were found to overlap with L15, L11/L16, L36/L37, S3, S10 and S33 from S. cerevisiae, respectively. Similar coelectrophoresis of E. coli 125I-labelled proteins with unlabelled rat liver and wheat germ ribosomal proteins showed the former to overlap with proteins L1, L11, L14, L16, L19, L20 and the latter with L2, L5, L6, L15, L17 from E. coli.     

Dephosphorylation of 40S ribosomal subunits by phosphoprotein phosphatase activity from rabbit reticulocytes.

Phosphoprotein phosphatase activities which remove phosphoryl groups from ribosomal protein have been partially purified from rabbit reticulocytes by chromatography on DEAE-cellulose. Two major peaks of phosphoprotein phosphatase activity were observed when 40S ribosomal subunits, phosphorylated $\text{in vitro}$ with cyclic AMP-regulated protein kinases and (γ-³²P)ATP, were used as substrate. The phosphatase activity eluting at 0.14 M KCl was characterized further using ribosomal subunits phosphorylated $\text{in situ}$ by incubation of intact reticulocytes with radioactive inorganic phosphate. Phosphate covalently bound to 40S ribosomal subunits and 80S ribosomes was removed by the phosphatase activity. The enzyme was not active with phosphorylated proteins associated with 60S ribosomal subunits.