Methylation of ribosomal proteins in Bacillus subtilis

We measured the methylation of ribosomal proteins from the 30S and 50S subunits of Bacillus subtilis after growing the cells in the presence of [1-14C]methionine and [methyl-3H]methionine. Two-dimensional polyacrylamide gel electrophoretic analysis revealed a preferential methylation of the 50S ribosomal proteins. Proteins L11 and L16, and possibly L9, L10, L18, and L20, were methylated. On the other hand, only two possibly methylated proteins were found on the 30S subunit. A comparison of these results with those for Escherichia coli suggests a common methylation pattern for the bacterial ribosomal proteins.     

Methylated amino acids in the proteins of the cytoplasmic ribosome of Tetrahymena thermophila

Two-dimensional electrophoretic analysis of proteins of the cytoplasmic ribosome of the protozoa Tetrahymena thermophila labeled in vivo with L-[14C1]methionine and L-[3H-methyl]methionine identifies one heavily methylated protein in each ribosomal subunit. These proteins, S31 and L21, each contain N epsilon-trimethyl-lysine.     

Methylation of Ribosomal Proteins in Escherichia coli

Escherichia coli was grown in a medium containing [1-(14)C]methionine and [methyl-(3)H]methionine, and the (3)H/(14)C ratio was determined for each of the ribosomal proteins derived from the 70S ribosome. Evidence indicates that six proteins from the 50S subunit were methylated: L7, L9, L11, L12, L18, and L33. Methylation of several other 50S proteins (such as L1, L3, L5, etc.) may also occur. The methylated amino acids in protein L11 have been characterized further and found to be predominately epsilon-trimethyllysine. A small amount of a compound tentatively identified as N(G), N'(G)-dimethylarginine was also detected.     

Subcellular distribution of ribosomal proteins in Dictyostelium discoideum

The distribution of ribosomal proteins in monosomes, polysomes, the postribosomal cytosol, and the nucleus was determined during steady-state growth in vegetative amoebae. A partitioning of previously reported cell-specific ribosomal proteins between monosomes and polysomes was observed. L18, one of the two unique proteins in amoeba ribosomes, was distributed equally among monosomes and polysomes. However S5, the other unique protein, was abundant in monosomes but barely visible in polysomes. Of the developmentally regulated proteins, D and S6 were detectable only in polysomes and S14 was more abundant in monosomes. The cytosol revealed no ribosomal proteins. On staining of the nuclear proteins with Coomassie blue, about 18, 7 from 40S subunit and 11 from 60S subunit, were identified as ribosomal proteins. By in vivo labeling of the proteins with [35S]methionine, 24 of the 34 small subunit proteins and 33 of the 42 large subunit proteins were localized in the nucleus. For the majority of the ribosomal proteins, the apparent relative stoichiometry was similar in nuclear preribosomal particles and in cytoplasmic ribosomes. However, in preribosomal particles the relative amount of four proteins (S11, S30, L7, and L10) was two- to four-fold higher and of eight proteins (S14, S15, S20, S34, L12, L27, L34, and L42) was two-to four-fold lower than that of cytoplasmic ribosomes.     

Preparation of Escherichia coli 70S ribosomes labeled with 35S

[35S]--70S ribosomes (150 Ci/mmol) were isolated from E. coli MRE-600 cells grown on glucose-mineral media in the presence of [35S] ammonium sulfate. The labeled 30S and 50S subunits were obtained from [35S] ribosomes by centrifugation in a sucrose density gradient of 10--30% under dissociating conditions (0.5 mM Mg2+). The activity of [35S]--70S ribosomes obtained by reassociation of the labeled subunits during poly(U)-dependent diphenylalanine synthesis was not less than 70%. The activity of [35S]--70S ribosomes during poly(U)-directed polyphenylalanine synthesis was nearly the same as that of the standard preparation of unlabeled ribosomes. The 23S, 16S and 5S RNAs isolated from labeled ribosomes as total rRNA contained no detectable amounts of their fragments as revealed by polyacrylamide gel electrophoresis. The [35S] ribosomal proteins isolated from labeled ribosomes were analyzed by two-dimensional gel electrophoresis. The [35S] label was found in all proteins, with the exception of L20, L24 and L33 which did not contain methionine or cysteine residues.     

Methylation of ribosomal proteins in bacteria: evidence of conserved modification of the eubacterial 50S subunit.

Methylation of the 50S ribosomal proteins from Bacillus stearothermophilus, Bacillus subtilis, Alteromonas espejiana, and Halobacterium cutirubrum was measured after the cells were grown in the presence of [1-14C]methionine or [methyl-3H]methionine or both. Two-dimensional polyacrylamide gel electrophoretic analysis revealed, in general, similar relative electrophoretic mobilities of the methylated proteins from each eubacterium studied. Proteins known to be structurally and functionally homologous in several microorganisms were all methylated. Thus, the following group of proteins, which appear to be involved in peptidyltransferase or in polyphenylalanine-synthesizing activity in B. stearothermophilus (P.E. Auron and S. R. Fahnestock, J. Biol. Chem. 256:10105-10110, 1981), were methylated (possible Escherichia coli methylated homologs are indicated in parentheses): BTL5(EL5), BTL6(EL3), BTL8(EL10), BTL11(EL11), BTL13(EL7L12) and BTL20b(EL16). In addition, the pentameric ribosomal complex BTL13 X BTL8, analogous to the complex EL7L12 X EL10 of E. coli, contained methylated proteins. Analysis of the methylated amino acids in the most heavily methylated proteins, BSL11 from B. subtilis and BTL11 from B. stearothermophilus, showed the presence of epsilon-N-trimethyllysine as the major methylated amino acid in both proteins, in agreement with known data for E. coli. In addition, BSL11 appeared to contain trimethylalanine, a characteristic, modified amino acid previously described only in EL11 from E. coli. These results and those previously obtained from other bacteria indicate a high degree of conservation for ribosomal protein methylation and suggest an important, albeit unknown, role for the modification of these components in eubacterial ribosomes.     

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.     

Structure of the yeast ribosomes. Proteins associated with the rRNA.

Polyamines have been shown to bind to doubled stranded regions of rRNA [3]. Therefore, ribosomal proteins that can be cross linked to these molecules in the ribosomes structure must be bound to or located in the vicinity of the RNA. This technique is the first to yield results on the proteins associated with the rRNA in the eukaryotic ribosome where the lack of purified ribosomal proteins does not allow the use of direct binding studies as in bacterial systems. Proteins S7, S10, S13, S21, S22 and S27 in the small subunit and L2/3, L5, L10/12, L19/20, L22, L23, L36/37, L42 and L43' in the large subunit are labelled when cross linked to [14C]spermidine using 1,5-difluoro 2,4-dinitrobenzene and are good candidates to be RNA-binding proteins in ribosomes from Saccharomyces cerevisiae.     

Induction of cell-specific ribosomal proteins in aggregation-competent nonmorphogenetic Dictyostelium discoideum

Vegetatively growing amoebae, if shaken in a starvation (nonnutrient) buffer, acquired aggregation competence, but do not embark on a morphogenetic program. The quantitative variation of ribosomal proteins in vegetative and aggregation-competent cells was compared by labeling the different cell types with [35S]methionine. Vegetative cells were examined at various phases of the growth cycle. No changes could be detected in the content of ribosomes or the apparent stoichiometry of ribosomal proteins in growing cells. In stationary phase cells, the net ribosome content declined to 15% of that observed in logarithmic phase, but the relative amounts of individual ribosomal proteins were not altered. Although aggregation-competent cells contained 30% less ribosomes compared with logarithmic phase cells, the total fraction of newly made ribosomal proteins was the same in both. In contrast to vegetative cells, distinct changes were induced in the ribosomal proteins of aggregation-competent cells. The composition of ribosomes in aggregation-competent phase resembled in every respect that observed in spore cells. As reported earlier, changes were found in all 12 of the developmentally regulated ribosomal proteins. For the majority of newly made ribosomal proteins during aggregation competence, the stoichiometry was similar to that in logarithmically growing cells. However, the relative synthesis of some was particularly higher (13- to 46-fold for A and L; 3- to 8-fold for D, E, S24, L3, S6, and L4) compared with logarithmic phase cells. About 18 proteins, which included the cell-specific ribosomal proteins L18, S10, S14, S16, and L11, were synthesized in lesser amounts than in logarithmic phase cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of 5SrRNA as a positive effector of some aminoacyl-tRNA synthetases in macromolecular complexes, with specific reference to methionyl-tRNA synthetase.

1) Rat liver 5SrRNA enhanced the activity of methionyl-tRNA synthetase in the macromolecular aminoacyl-tRNA synthetase complex (Fraction B) purified from a rat liver supernatant. 5SrRNA-L5 protein complexes (5SrRNP) had similar effects, whereas other ribosomal RNAs and E. coli 5SrRNA had no effect. 2) 5SrRNA increased the activity of the complex for methionine-dependent ATP-PPi exchange. 3) 5SrRNA increased the activities of methionyl-, arginyl-, and isoleucyl-tRNA synthetases in the complex, but scarcely affected its leucyl-, lysyl-, and glutamyl-tRNA synthetase activities. 4) 5SrRNA increased the activities of the rat liver supernatant for the attachment of [35S]methionine, [3H]isoleucine, [3H]lysine, [3H]proline, [3H]threonine, [3H]tyrosine, and [3H]phenylalanine to endogenous tRNA markedly, and those for [3H]leucine, [3H]arginine, [3H]aspartic acid, and [3H]histidine slightly, but did not affect those for [3H]glutamic acid, [3H]glycine, [3H]valine, [3H]alanine, and [3H]tryptophan. 5) Preincubation of the rat liver supernatant with an antibody against Artemia salina ribosomal protein L5, that cross-reacted with the rat liver ribosomal protein L5, decreased the attachment of [35S]methionine and [3H]isoleucine to endogenous tRNA, and 5SrRNA and 5SRNP enhanced these activities of the supernatant preincubated with antibody. On the other hand, the antibody did not affect that for [3H]alanine. Immune dot blot analysis using the antibody against L5 showed the presence of immunologically the same protein as L5 in the liver supernatant. Northern blot analysis of RNA in the immunoprecipitate prepared from the liver supernatant incubated with the antibody against L5 indicated that 5SrRNA was complexed with L5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the biosynthesis of sulfolipids in the Diatom Nitzschia alba.

Labeling of sulfolipids in Nitzschia alba was studied after growth of the cells in media containing L-[35S]cystine, L-[35S], L-[35S]cysteine, L-[35S]-methionine or a mixture of L-[Me-3H]methionine and L-[35S]methionine, [35S]Cysteine or [35S]cystine labeled the deoxyceramide sulfonate and the sulfonium analog, phosphatidylsulfocholine (and its lyso derivative) but not the sterol sulfate nor the sulfoquinovosyl diglyceride; [35S]methionine labeled only the phosphatidylsulfocholine and its lyso derivative. With the [35S]- and [Me-3H]methionine mixture (3H/35S ratio 1.0) the phosphatidylsulfocholine had a 3H/35 S ratio of 1.5 indicating that both sulfonium methyl groups were derived from methionine. Probable biosynthetic pathways for these novel sulfolipids are discussed.     

Identification and analysis of three myristylated vaccinia virus late proteins.

Previous studies have shown that at least three vaccinia virus (VV) late proteins (with apparent molecular asses of 37, 35, and 25 kDa) label with myristic acid. Time course labeling of VV-infected cells with [3H]myristic acid reveals at least three additional putative myristylproteins, with apparent molecular masses of 92, 17, and 14 kDa. The 25-kDa protein has previously been identified as that encoded by the L1R open reading frame, leaving the identities of the remaining proteins to be determined. Sequence analysis led to the preliminary identification of the 37-, 35-, and 17-kDa proteins as G9R, A16L, and E7R, respectively. Using synthetic oligonucleotides and PCR techniques, each of these open reading frames was amplified by using VV DNA as a template and then cloned individually into expression vectors behind T7 promoters. These plasmid constructs were then transcribed in vitro, and the resulting mRNAs were translated in wheat germ extracts and radiolabeled with either [35S]methionine or [3H]myristic acid. Each wild-type polypeptide was labeled with [35S]methionine or [3H]myristic acid in the translation reactions, while mutants containing an alanine in place of glycine at the N terminus were labeled only with [35S]methionine, not with myristic acid. This result provided strong evidence that the open reading frames had been correctly identified and that each protein is myristylated on a glycine residue adjacent to the initiating methionine. Subcellular fractionations of VV-infected cells suggested that A16L and E7R are soluble, in contrast to L1R, which is a membrane-associated protein.     

Coordinate control of syntheses of ribosomal ribonucleic acid and ribosomal proteins during nutritional shift-up in Saccharomyces cerevisiae.

We investigated the regulation of ribosome synthesis in Saccharomyces cerevisiae growing at different rates and in response to a growth stimulus. The ribosome content and the rates of synthesis of ribosomal ribonucleic acid and of ribosomal proteins were compared in cultures growing in minimal medium with either glucose or ethanol as a carbon source. The results demonstrated that ribosome content is proportional to growth rate. Moreover, these steady-state concentrations are regulated at the level of synthesis of ribosomal precursor ribonucleic acid and of ribosomal proteins. When cultures growing on ethanol were enriched with glucose, the rate of ribosomal ribonucleic acid synthesis, measured by pulsing cells with [methyl-3H]methionine, increased by 40% within 5 min, doubled within 15 min, and reached a steady state characteristic of the new growth medium by 30 min. Labeling with [3H]leucine reveal a coordinate increase in the rate of synthesis of 30 or more ribosomal proteins as compared with that of total cellular proteins. Their synthesis was stimulated approximately 2.5-fold within 15 min and nearly 4-fold within 60 min. The data suggest that S. cerevisiae responds to a growth stimulus by preferential stimulation of the synthesis of ribosomal ribonucleic acid and ribosomal proteins.     

Phosphorylation in vitro and in vivo of ribosomal proteins from Saccharomyces cerevisia.

Crude ribosomes from Saccharomyces cerevisiae cultures were phosphorylated in vitro when incubated in the presence of [gamma-32P]ATP. Analysis of the ribosomal proteins with two-dimensional electrophoresis revealed that of the 29 proteins identified in the small subunit, only protein S6 was phosphorylated. Of the 37 proteins identified in the large subunit, one was highly phosphorylated (L3) and two only slightly phosphorylated (L11 and L14). The protein kinase activity associated with the ribosomes was extracted with 1 M KCl and was not dependent on adenosine 3':5'-monophosphate; it preferentially phosphorylated casein and phosvitin, but was less active on histones. Structural ribosomal proteins were also phosphorylated in vivo when the yeast cultures were incubated with [32P]orthophosphate; the radioactivity resistant to hydrolysis by hot perchloric acid was incorporated into the proteins of the two subunits. Radioactive phosphoserine was found by subjecting hydrolysates of ribosomal proteins to high-voltage electrophoresis. After two-dimensional electrophoresis, one poorly phosphorylated protein (S10) was identified in the small subunit. In the large subunit, one protein (L3) was highly labelled, and two proteins (L11 and L24) only slightly labelled.     

Ribosome topography in baby hamster kidney cells infected with Sindbis and vesicular stomatitis viruses

The topography of polysomal ribosomes in mock-infected and in Sindbis virus- and vesicular stomatitis virus-infected BHK cells was investigated using a double, radioactive labelling technique. Ribosomal proteins in intact polysomes were surface labelled by reductive methylation using [14C]formaldehyde. Following removal of ribosomal RNA, proteins were denatured in 6 M guanidine and labelled with [3H]borohydride. Labelled ribosomal proteins were separated by electrophoresis in two-dimensional gels and the 3H/14C ratio for each ribosomal protein was taken as an index of its relative surface exposure in intact ribosomes. Comparison of the ratios for individual ribosomal proteins in Sindbis virus-infected vs. control polysomes indicated that proteins L7, L8, L17, L26 and S19 became more 'buried' and others such as L4, L29, L36, S2 and S26 became more 'exposed' in infected cells. Most of the topographical alterations occurred in the large ribosomal subunit. In contrast, infection of BHK cells with vesicular stomatitis virus induced little or no topographical alteration.     

Phosphorylation of multiple proteins of both ribosomal subunits in rat cerebral cortex in vivo. Effect of adenosine 3'':5''-cyclic monophosphate.

Investigations were carried out on the phosphorylation of ribosomal proteins in vivo in cerebral cortices of immature rats. Two-dimensional electrophoresis revealed that the cerebral 40S subunit contained at least four ribosomal proteins which were phosphorylated in animals given [32P]orthophosphate intracisternally. These proteins exhibited electrophoretic properties similar to those of the constitutive basic proteins S2, S3a, S5 and S6. The cerebral 60S subunit contained several proteins that were phosphorylated in vivo, including three basic proteins with electrophoretic mobilities similar to those of ribosomal proteins L6, L14 and L19. Four other proteins associated with the 60S subunit that were more acidic were also phosphorylated. Phosphorylated congeners of 40S and 60S ribosomal proteins could often be detected in distinct protein-stained spots on two-dimensional electrophoretograms. The cerebral S6 protein consisted of at least five distinct species in different states of phosphorylation. Administration of N6O-2' dibutyryl cyclic AMP increased the proportion of the more phosphorylated congeners of the S6 protein, but appeared to have little or no effect on phosphorylation of other cerebral ribosomal proteins. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine also stimulated S6-protein phosphorylation; N2O2'-dibutyryl cyclic GMP had no effect on this process. These observations indicate that several ribosomal proteins of both subunits are normally phosphorylated in rat cerebral cortex in situ. The results also suggest that selective and specific alterations in the phosphorylation state of the S6 ribosomal protein of the cerebral 40S subunit may accompany the production of cyclic AMP during neural activation.     

Proteins of animal ribosomes. XXV. Proteins of rat liver ribosomes protected by polyuridylic acid from methyl acetimidate substitution.

Addition of poly(U) to complexes of 40S and 60S subunits of rat liver ribosomes decreases the substitution of amino groups of 12 proteins of the small ribosomal subunit and of 11 proteins of the large subunit by [14C]-methyl acetimidate. When comparing the results obtained with this amino group specific reagent with the reactivity of the proteins against iodoacetamide it becomes obvious that 4 proteins of the small ribosomal subunit (S12, 18, 19, 24) and 3 proteins of the large one (L20, 22, 25) are partially protected by poly(U) against reaction with both reagents.     

The effect of magnesium starvation on the dissociation of ribosomal proteins from Escherichia coli K-12 ribosomes.

The effect of magnesium starvation upon the fate of individual ribosomal proteins was studied in Escherichia coli. During a 21 h incubation in the absence of Mg2+ the 30 S subunit was more susceptible to degradation, retaining an average 31.9% of its ribosomal proteins as compared to 40.0% for the 50 S subunit. An examination of those 50-S proteins dissociated to a lesser extent than the average value (L1, L2, L3, L7, L10, L13, L16, L17, L19, L21, L22, L23, and L29) revealed that, with the exception of L16, all were classified by Dohme and Nierhaus [5] as tightly bound. Of the ribosomal proteins dissocated during magnesium starvation only five were reincorporated (and these to a minimal degree) during recovery of cells in a medium containing Mg2+. These studies suggest that ribosomal proteins once released from the ribosome particles during magnesium starvation are not reutilized in the assembly of new subunits.