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PubMed1996

Phosphorylation of ribosomal proteins by ribosome-associated protein kinases of Trichosporon cutaneum.

Four ribosomal proteins of Mr 13 kDa, 15 kDa, 19 kDa and 38 kDa were identified as phosphorylation substrates for protein kinases tightly associated with Trichosporon cutaneum ribosomes. It was found that proteins of 13 kDa, 19 kDa and 38 kDa were phosphorylated by multifunctional casein kinase II while the protein of 15 kDa by casein kinase I. Proteins of 13 kDa and 38 kDa were detected in the large subunits while 15 kDa and 19 kDa in the small ribosomal subunits. By using isoelectrofocusing the protein of 13 kDa was resolved into two individual phosphorylated forms. The phosphorylation level of both forms was much higher in ribosomes from the cells collected at the exponential growth phase than in those from the stationary phase. The same phosphoprotein forms were identified in ribosomes from in vitro and in vivo [32P]-labelling experiments.

Casein Kinase II↗
PubMed1979

A missense mutation in the gene coding for ribosomal protein S17 (rpsQ) leading to ribosomal assembly defectivity in Escherichia coli.

The conditionally lethal mutation, 2861 mis, has been mapped inside the ribosomal protein gene cluster at 72 minutes on the Escherichia coli chromosome and was found to cotransduce at 97% with rpsE (S5). The 2861 mis mutation leads to thermosensitivity and impaired assembly in vivo of 30S ribosomal particles at 42 degrees C. The strain carrying the mutation has an altered S 17 ribosomal protein; the mutational alteration involves a replacement of serine by phenylalanine in protein S 17. Spontaneous reversion to temperature independence can restore the normal assembly in vivo of 30 S ribosomal subunits at 42 degrees C and the normal chromatographical behaviour of the S 17 ribosomal protein in vitro. We conclude therefore that the 2861 mis mutation affects the structural gene for protein S 17 (rpsQ).

Bacterial Proteins↗
PubMed1983

Yeast ribosomal proteins: VII. Cytoplasmic ribosomal proteins from Schizosaccharomyces pombe.

The cytoplasmic ribosomal proteins from a fission yeast Schizosaccharomyces pombe were analysed by two-dimensional polyacrylamide gel electrophoresis. Seventy-three protein species were identified in the 80S ribosome, and named SP-S1 to SP-S33 and SP-L1 to SP-L40 in the small and large subunits, respectively. Many of these proteins could be correlated to those of Saccharomyces cerevisiae on the basis of their electrophoretic mobilities. Eleven proteins were isolated from the 80S ribosome, and their amino acid compositions were determined. Of these, SP-S6, SP-L1, SP-L12, SP-L15, SP-L17, SP-L27, SP-L36 and SP-L40c and d were sequenced from their amino-termini. SP-S28 and SP-L2 appear to have their amino-termini blocked. These results were compared with the data available for the S. cerevisiae and rat liver ribosomal proteins. The S. cerevisiae counterparts of the eight proteins mentioned above were found to be YS4, YL1, YL10, YL14, YL35, YL40 and YL44c and d, respectively. The rat liver counterparts of SP-S6, SP-L1, SP-L27 and SP-L40c and d were the rat S6, L4, L37 and P2, respectively. Comparison of the partial sequences of these ribosomal proteins suggests that these two yeasts are relatively far apart, phylogenetically.

Amino Acid Sequence↗
PubMed1975

Ribosomal and non-ribosomal RNA synthesis in vitro.

The synthesis of total and ribosomal RNA using nucleoids of Escherichia coli as template was measured; of the total RNA synthesized by endogenous RNA polymerase which only completes chains, and added RNA polymerase which initiates new chains, 50-70 and 3-5%, repectively, was rRNA. Total RNA synthesis by added enzyme, however, was 10-20 times higher than endogenous RNA synthesis; thus rRNA was synthesized at the same rate by the endogenous and the added enzyme. We conclude that the percentage rRNA in vitro is no measure of the rate of rRNA synthesis. Furthermore, it follows that the added enzyme, like the endogenous one, is packed at the physical limit on the ribosomal cistrons. Consequently, initiation of ribosomal cistrons by added enzyme was at or near the maximal rate possible for this system in which the elongation rate is 10-20% of that in vitro. When RNA synthesis was assayed at various ratios of RNA polymerase to phenol-extracted DNA, the amount of rRNA made per DNA, which is a measure of the frequency of transcription of ribosomal cistrons, varied. The ratio of rRNA synthesis relative to total RNA synthesis also varied, but in a different way, again leading to the conclusion that this ratio, as determined in vitro, does not reflect the efficiency of transcription of the ribosomal cistrons.

DNA↗
PubMed1976

Effect of nitrofurazone on bacterial RNA and ribosome synthesis and on the function of ribosomes.

Exposure of E. coli B/r to nitrofurazone strongly inhibits the synthesis of all classes of RNA and both ribosomal sub-units. Polysome formation is likewise inhibited. However, in E. coli nfr-207 a mutant of B/r which lacks nitrofurazone-reductase I, the synthesis of RNA, ribosomal sub-units and formation of polysomes are not significantly affected. This result implies that a reduced metabolite of the drug rather than the drug itself is the active agent. The ability of ribosomes isolated from nitrofurazone-treated E. coli B/r to carry out poly-U directed polyphenylalanine synthesis was lower than that of ribosomes from untreated cells. 14C from labelled nitrofurazone was found to bind to ribosomal sub-units.

DNA-Directed RNA Polymerases↗
PubMed1993

Synthetic peptides and ribosomal proteins as substrate for 60S ribosomal protein kinase from yeast cells.

Kinetic studies on the 60S protein kinase were conducted with synthetic peptides and ribosomal proteins as substrate. Peptide RRREEESDDD proved to be the best synthetic substrate for this enzyme. The peptide has a sequence of amino acids which most closely resembles the structure of potential phosphorylation sites in natural substrates, i.e., acidic ribosomal proteins. The superiority of certain kinetic parameters for 60S kinase obtained with the native whole 80S ribosomes over those of the isolated fraction of acidic ribosomal proteins indicates that the affinity of 60S kinase to the specific protein substrate not only depends on the structure of the polypeptide chain around the target amino acid but also on its native structure within the 80S ribosome.

Amino Acid Sequence↗
PubMedDate not supplied

Polysaccharide nature of O antigen in protective ribosomal preparations from Shigella: experimental evidence and implications for the ribosomal vaccine concept.

Shigella ribosomal vaccine (SRV) was previously shown to be highly active in induction of mucosal and systemic O-antibody response and protection against Shigella infection in guinea pigs and monkeys. In this study, the O-specific component (OSC) was isolated from the SRV by affinity chromatography using rabbit O antibodies coupled to CNBr-Sepharose. The results of the reaction with carbocyanine dye as well as chemical data show that ribosomal OSC is devoid of lipid A and KDO, which are characteristic of classical LPS. The comparison of OSC with various LPS-related substances led to the conclusion that ribosomal OSC is similar to and probably identical with cytoplasmic O polysaccharide (L hapten), an O-side-chain polymer which accumulates in cytoplasm. It is hypothesized that the extremely high immunogenicity of SRV depends on a cooperative action of OSC, representing an epitope-specific part of the vaccine, and a ribosomal particle which serves as a vector, providing amplification of the immunogenic effect. The data obtained indicate the presence of a non-covalent link between the two components of the ribosomal vaccine.

Antigens, Bacterial↗
PubMed2005

ERj1p uses a universal ribosomal adaptor site to coordinate the 80S ribosome at the membrane.

Ribosomes translating secretory and membrane proteins are targeted to the endoplasmic reticulum membrane and attach to the protein-conducting channel and ribosome-associated membrane proteins (RAMPs). Recently, a new RAMP, ERj1p, has been identified that recruits BiP to ribosomes and regulates translational activity. Here we present the cryo-EM structure of a ribosome-ERj1p complex, revealing how ERj1p coordinates the ribosome at the membrane and how allosteric effects may mediate ERj1p's regulatory activity.

Animals↗
PubMed1965

The distribution of ribosomal ribonucleic acids among subcellular fractions from bacteria and the adverse effect of the membrane fraction on the stability of ribosomes.

1. The distributions of nucleic acids and protein among fractions obtained by differential centrifugation from species of Pseudomonas, Aerobacter, Escherichia, Proteus and Bacillus have been studied. 2. The DNA in a cell wall-membrane fraction obtained by low-speed centrifugation from the Gram-negative species could be removed by homogenizing and subsequent washing. About 7-14% of the total RNA remained firmly attached and resembled ribosomal RNA in base composition. A similar fraction from the Gram-positive B. subtilis contained about one-half of the total bacterial DNA and only 60% of this could be removed by homogenizing and subsequent washing. 3. A deposit obtained by high-speed centrifugation could be separated into a heavy ribosome layer and a light turbid layer. In E. coli B the latter contained about equal concentrations of RNA and DNA and accounted for about one-half of the total bacterial NADP-activated 6-phosphogluconate dehydrogenase. 4. The washed cell wall-membrane fraction from most species accelerated the degradation of ribosomes. In Pr. vulgaris the activity of this fraction was exceptionally high and resulted in the progressive degradation of ribosomes during their isolation from this species. 5. A possible connexion between ribosome degradation and the synthesis of flagella is discussed in the light of these results.

Bacillus subtilis↗
PubMed2001

A novel site of antibiotic action in the ribosome: interaction of evernimicin with the large ribosomal subunit.

Evernimicin (Evn), an oligosaccharide antibiotic, interacts with the large ribosomal subunit and inhibits bacterial protein synthesis. RNA probing demonstrated that the drug protects a specific set of nucleotides in the loops of hairpins 89 and 91 of 23S rRNA in bacterial and archaeal ribosomes. Spontaneous Evn-resistant mutants of Halobacterium halobium contained mutations in hairpins 89 and 91 of 23S rRNA. In the ribosome tertiary structure, rRNA residues involved in interaction with the drug form a tight cluster that delineates the drug-binding site. Resistance mutations in the bacterial ribosomal protein L16, which is shown to be homologous to archaeal protein L10e, cluster to the same region as the rRNA mutations. The Evn-binding site overlaps with the binding site of initiation factor 2. Evn inhibits activity of initiation factor 2 in vitro, suggesting that the drug interferes with formation of the 70S initiation complex. The site of Evn binding and its mode of action are distinct from other ribosome-targeted antibiotics. This antibiotic target site can potentially be used for the development of new antibacterial drugs.

Aminoglycosides↗
PubMed1974

Coordinate inhibition of elongation factor G function and ribosomal subunit association by antibodies to several ribosomal proteins.

We previously showed that treatment of Escherichia coli ribosomes with antibodies specific for proteins L7 and L12 inhibits both EF-G.GDP binding and ribosome-dependent GTP hydrolysis (Highland et al. (1973) Proc. Nat. Acad. Sci. USA 70, 142-150; and Kischa et al. (1971) Nature New Biol. 233, 62-63). We now report that antibodies to six additional proteins also inhibit GTP hydrolysis, but do not inhibit EF-G.GDP binding. Moreover, inhibition by these antibodies is dependent on the state of association of the treated ribosomes. When 70S couples are treated, only antibodies to proteins L14 and L23 are inhibitory and then only partially. However, when separated ribosomal subunits are treated individually and then mixed with the complementary untreated subunits, inhibition by antibodies L14 and L23 is complete, and antibodies to proteins L19, L27, S9, and S11 now show an inhibitory effect. In addition, treatment of subunits with any of these six antibodies (but not those to L7 or L12) results in inhibition of reassociation, which is presumably responsible for the inhibition of hydrolysis. These data suggest that the area of interaction between EF-G and the ribosome is restricted to proteins L7 and L12, and that antibodies to proteins L14, L19, L23, L27, S9, and S11, but not L7 and L12, block the physical association of the subunits.

Antibodies↗
PubMed1998

A ribosomal protein is required for translational regulation of GCN4 mRNA. Evidence for involvement of the ribosome in eIF2 recycling.

In amino acid-starved yeast cells, inhibition of the guanine nucleotide exchange factor eIF2B by phosphorylated translation initiation factor 2 results in increased translation of GCN4 mRNA. We isolated a suppressor of a mutant eIF2B. The suppressor prevents efficient GCN4 mRNA translation due to inactivation of the small ribosomal subunit protein Rps31 and results in low amounts of mutant 40 S ribosomal subunits. Deletion of one of two genes encoding ribosomal protein Rps17 also reduces the amounts of 40 S subunits but does not suppress eIF2B mutations or prevent efficient GCN4 translation. Our findings show that Rps31-deficient ribosomes are altered in a way that decreases the eIF2B requirement and that the small ribosomal subunit mediates the effects of low eIF2B activity on cell viability and translational regulation in response to eIF2 phosphorylation.

Alleles↗
PubMed1983

Quantitation of cation binding to wheat germ ribosomes: influences on subunit association equilibria and ribosome activity.

The binding of Mg2+, spermine, and spermidine to wheat germ ribosomes was quantitated following equilibrium dialysis. The Mg2+ binding data demonstrate that Mg2+ and K+ compete for binding to the ribosomes. Mg2+ binding saturates at approximately 0.56 positive charges per phosphate (+/P). The Mg2+, spermine and spermidine binding data indicate that either polyamine replaces Mg2+ upon binding to the ribosomes. Mg2+ and polyamine binding combined saturates at approximately 0.29 +/P under the conditions reported. When a critical number of Mg2+ ions are replaced by either polyamine, the activity of the ribosomes falls dramatically. Ribosomal subunit association increases with the degree of phosphate charge neutralization due to the binding of Mg2+. Total charge neutralization during subunit association by Mg2+ and polyamine binding combined, is much less than that achieved by Mg2+ alone.

Cations↗
PubMed1986

The gene for Spirodela oligorhiza chloroplast ribosomal protein homologous to E. coli ribosomal protein L16 is split by a large intron near its 5' end: structure and expression.

The nucleotide sequence of a Spirodela chloroplast DNA fragment, which directs the synthesis of a approximately 15 kD chloroplast ribosomal protein in an E. coli cell free system, has been determined. The deduced aminoacid sequence of the open reading frame shows extensive homology with E. coli ribosomal protein L16. Primer extension analysis, S1 nuclease mapping and nucleotide sequence analysis indicate that the chloroplast L16 gene (rpl16) is interrupted by a 1411 bp intron, which separates a short 5' exon from a large 3' exon. The shorter in vitro synthesized ribosomal protein results from an artificial initiation event at an internal ATG codon in the 3' exon. The sequences at the 5' and 3' splice sites of the intron are similar to consensus sequences described for other, class II intron containing, protein coding chloroplast genes. Northern hybridization experiments reveal 6 stable transcripts of rpl16 ranging from 500 b to greater than 4000 b. As determined by S1 nuclease mapping, the 3'-end of the smallest transcript maps exactly after the stem of a proposed termination signal. Finally, the implications of the finding of a cluster of several chloroplast ribosomal protein genes and possible polycistronic transcription of this chloroplast DNA region, are discussed in relation to the organization and expression of ribosomal protein genes found in the S10 operon of E. coli.

Amino Acid Sequence↗
PubMed1974

Pleiotropic antibiotic resistance mutations associated with ribosomes and ribosomal subunits in Mycobacterium smegmatis.

Viomycin-resistant strains isolated from Mycobacterium smegmatis demonstrated pleiotropic resistance to tuberactinomycin-N, capreomycin, streptomycin, and kanamycin as a result of mutational alteration of ribosomes, even though they were selected for resistance to a single antibiotic. The pleiotropic drug resistance of three mutants isolated by stepwise selection for resistance to viomycin was due to alteration of the 30S ribosomal subunit. One mutant, strain A, isolated independently by multiple-step selection to viomycin resistance, was resistant to viomycin, tuberactinomycin-N, and capreomycin through an alteration of the 50S ribosomal subunit, whereas it was sensitive to kanamycin but resistant to streptomycin through an alteration of the 30S ribosomal subunit. Three streptomycin-resistant strains, which were isolated by one-step selection at a high concentration of streptomycin, did not show significant co-resistance to any other antibiotics tested in culture and cell-free systems; streptomycin resistance in these mutants was localized on the 30S ribosomal subunit.

Drug Resistance, Bacterial↗
PubMed1975

Effects of dihydrostreptomycin on ribosome function in vivo: lack of correlation between changes in ribosome patterns and growth.

In vivo treatment of susceptible Escherichia coli cultures with low concentrations of dihydrostreptomycin leads to a decline in polysomes and a corresponding increase in 70S particles which behave as run-off ribosomes, as well as free 30S and 50S subunits. We have examined the timing and extent of these effects on ribosomes and compared them to the effects of this antibiotic on growth and protein synthesis. We have shown that no changes in ribosome distribution are observed until growth inhibition by dihydrostreptomycin is almost complete. Thus, intracellular dihydrostreptomycin can inhibit growth and net protein synthesis without apparently affecting the ribosome cycle. Since it is known that the antibiotic combines with free 30S subunits, the question is how such combination can bring about the observed inhibition of protein synthesis and growth. We suggest that specific interaction of intracellular antibiotic with proteins of the 30S subunits allows repeated use of the ribosome cycle by such affected particles, but with selective misreading of certain amino acid codons as terminator codons, so that they produce incomplete polypeptide chains. The cumulative effect of such a mechanism would lead to eventual cessation of protein synthesis and growth.

Dihydrostreptomycin Sulfate↗
PubMed1979

Selective spin-labeling of the ribosomal proteins of 70S ribosomes from Escherichia coli.

We have used a series of N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl) maleimide spin labels of different length to label, covalently and selectively, the most reactive sulfhydryl groups of 70S ribosomal proteins of Escherichia coli. Under short periods of labeling (1--2 min), less than two spin labels per ribosome are incorporated and were shown to be distributed mainly on five ribosomal proteins in the following order: S18 greater than S21, L27 greater than S17, and S12. With a long period of labeling (3 h) up to 13 spin labels are attached to the ribosome, and protein S1 is the most labeled. The shape of the electron paramagnetic resonance (epr) signal shows two components with a predominance for the strongly immobilized orientation, and the percentage of these components in each spectra has been evaluated. When the distance between the nitroxide group and the maleimide-attaching group exceeds 6 A (1 A = 0.1 nm) the strongly immobilized orientation disappears. The effect of magnesium ions on these selectively spinlabeled ribosomes shows that the dissociation into subunits does not affect the epr signal, but more spin labels are incorporated into the subunits if labeling is performed under conditions of dissociation.

Bacterial Proteins↗
PubMed1981

A GTPase reaction accompanying the rejection of Leu-tRNA2 by UUU-programmed ribosomes. Proofreading of the codon-anticodon interaction by ribosomes.

The characteristics of a GTPase reaction between poly(U)-programmed ribosomes, EFTu . GTP, and the near-cognate aminoacyl (aa)-tRNA, Leu-tRNA Leu 2, have been studied to assess the role of this reaction in proofreading of the codon-anticodon interaction. The reaction resembles the GTPase reaction with cognate aa-tRNAs and EFTu . GTP in its substrate requirements, in its involving EFTu . GTP . aa-tRNA ternary complexes, and in its requiring a free ribosomal A-site. The noncognate reaction differs from the cognate one in that aa-tRNA becomes stably bound to the ribosomes only 5% of the time; it therefore seems best characterized as an abortive enzymatic binding reaction. The rate of reaction is a significant fraction (4%) of that of the cognate aa-tRNA, indicating that recognition of ternary complexes by ribosomes involves a level of error greater than that of translation as a whole. The rejection of the noncognate aa-tRNA following GTP hydrolysis is therefore a vital step in the translation process and fulfills the criteria set for a proofreading reaction. Leu-tRNA Leu 2 which escapes rejection through proofreading, forms a stable complex with the ribosomal A-site, so it appears that the Leu-tRNA2 which was rejected never reached the A-site and that proofreading precedes full A-site binding.

Anticodon↗