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PubMed1987

Characterization of a novel acidic protein of 38 kDa, A0, in yeast ribosomes which immunologically cross-reacts with the 13 kDa acidic ribosomal proteins, A1/A2.

A new ribosomal protein of 38 kDa, named A0, was detected in yeast ribosomes on immunoblotting. The antibody used here was that against A1/A2, 13 kDa acidic ribosomal proteins which cross-reacted with A0. Although A0 and A1/A2 share common antigenic determinants, they differ in the following biochemical properties. While A1/A2 could be extracted from ribosomes with ethanol and ammonium sulfate, A0 could not. A0 gave two protein spots in a less acidic region than for A1/A2 on two-dimensional gel electrophoresis. The heterogeneity observed for A0 was ascribable to phosphorylation because one spot disappeared after treatment of the ribosomes with phosphatase. The syntheses of A0 and A1/A2 are directed by different mRNA species, as judged with a cell-free translation system, ruling out the possibility that A0 is a precursor of A1/A2. Although a mammalian ribosomal protein equivalent to A0 has been shown to be associated with 13 kDa acidic proteins in the cytoplasm, essentially no A0 was detected on immunoblotting in the yeast cytosol, while a small but detectable amount of A1/A2 was present. The possibility that A0 is a eukaryotic equivalent of L10 of Escherichia coli is discussed.

Cytosol↗

PubMed1987

Structural and immunochemical characterization of a ribosomal protein from gram-positive Micrococcus luteus which is functionally homologous to Escherichia coli ribosomal protein S1.

Ribosomes from gram-positive Micrococcus luteus contain an acidic protein (ML-S1). ML-S1 has been purified by chromatography of ribosomes on a poly(U)-Sepharose column and the purified protein has a mobility in sodium dodecyl sulphate/polyacrylamide gels similar to that of ribosomal protein S1 of Escherichia coli (apparent Mr 72,000). Protein ML-S1 reacted with E. coli anti-S1 serum with an immunological partial-identity reaction. ML-S1 also reacted with antibodies raised against two structural domains of E. coli S1 (the N-terminal ribosome-binding domain and central and C-terminal nucleic-acid-binding domain). Weak reaction with antiserum to the nucleic-acid-binding domain of E. coli S1 was observed. ML-S1 was digested with trypsin under mild and exhaustive conditions. Mild digestion resulted in the production of a trypsin-resistant core (ML-S1F1) like E. coli S1. The fragment pattern obtained after exhaustive digestion differed appreciably from that obtained with E. coli S1. ML-S1 bound to poly(U) as strongly as E. coli S1 and also showed appreciable binding to denatured DNA. Addition of ML-S1 to S1-depleted ribosomes from E. coli and M. luteus markedly stimulated the poly(U)-directed polyphenylalanine synthesis. Phage MS2-RNA-dependent translation was also found to be stimulated by ML-S1 although to a much lesser extent than the stimulation by E. coli S1. At a molar excess of ML-S1 to ribosomes the protein showed a similar inhibitory effect to E. coli S1 on polypeptide synthesis. Our data indicate that ML-S1 retained the structural domains important for its function despite certain structural differences from E. coli S1.

Cross Reactions↗

PubMed2001

A ribosomal ATPase is a target for hygromycin B inhibition on Escherichia coli ribosomes.

We demonstrate that the transfer of fully charged aminoacyl-tRNAs into peptides directed by the MS2 RNA template requires both ATP and GTP, initiation factors (IF1, IF2, and IF3), elongation factors (EF-Tu, EF-Ts, and EF-G), and the ribosomal ATPase (RbbA). The nonhydrolyzable analogue AMPPCP inhibits the reactions, suggesting that hydrolysis of ATP is required for synthesis. The RbbA protein occurs bound to ribosomes and stimulates the ATPase activity of Escherichia coli 70S and 30S particles. The gene encoding RbbA harbors four ATP binding domains; the C-terminal half of the protein bears extensive sequence similarity to EF-3, a ribosome-dependent ATPase. Here, we show that the antibiotic hygromycin B selectively inhibits the ATPase activity of RbbA. Other antibiotics with similar effects on miscoding, streptomycin and neomycin, as well as antibiotics that impair peptide bond synthesis and translocation, had little effect on the ATPase activity of RbbA on 70S ribosomes. Immunoblot analysis indicates that at physiological concentrations, hygromycin B selectively releases RbbA from 70S ribosomes. Hygromycin B protects G1494 and A1408 in the decoding region, and RbbA enhances the reactivity of A889 and G890 of the 16S rRNA switch helix region. Cross-linking and X-ray diffraction data have revealed that this helix switch and the decoding region are in close proximity. Mutations in the switch helix (889-890) region affect translational fidelity and translocation. The binding site of hygromycin B and its known dual effect on the fidelity of decoding and translocation suggest a model for the action of this drug on ribosomes.

Adenosine Triphosphatases↗

PubMed1974

Neurospora crassa cytoplasmic ribosomes: isolation and characterization of a cold-sensitive mutant defective in ribosome biosynthesis.

Twenty-seven cold-sensitive mutants of Neurospora crassa were isolated by mutagenesis of wild-type conidia followed by filtration enrichment in complete medium at the nonpermissive temperature (10 C). Zone sedimentation analyses of cytoplasmic ribosomes isolated from the wild-type strain and from 14 of the mutant strains grown at 10 C indicate that one cold-sensitive mutant is defective in ribosome biosynthesis at that temperature: instead of the 2.3:1 mass ratio of 60S:37S ribosomal subunits characteristic of wild type, the mutant strain PJ30201 (called crib-1 for cytoplasmic ribosome biosynthesis) exhibits a mass ratio of approximately 7.2:1. Ribosomal subunits synthesized by strain PJ30201 at 25 C are present in wild-type proportions. The cold-sensitive and ribosomal phenotypes segregate together in tetrads isolated from crosses between strain PJ30201 and the wild type indicating that a single nuclear gene mutation is probably responsible for both mutant phenotypes. The crib-1 locus lies near the centromere in linkage group IV.

Centrifugation, Density Gradient↗

PubMed1998

Ribosomal protection from tetracycline mediated by Tet(O): Tet(O) interaction with ribosomes is GTP-dependent.

Tet(O) mediates tetracycline resistance by protecting the ribosome from inhibition. A recombinant Tet(O) protein with a histidine tag was purified and its activity in protein synthesis characterized. Tetracycline inhibited the rate of poly(Phe) synthesis, producing short peptide chains. Tet(O)-His was able to restore the elongation rate and processivity. 70S ribosomes bound tetracycline with high affinity. Tet(O)-His in the presence of GTP, but not GDP or GMP, reduced the affinity of the ribosomes for tetracycline. Non-hydrolyzable GTP analogs in the presence of the factor were also able to interfere with tetracycline binding. Ribosomes increased the affinity of Tet(O)-His for GTPgammaS. Tet(O), 70S ribosomes and GTPgammaS formed a complex that could be isolated by gel filtration. The GTP conformer is the active form of Tet(O) that interacts with the ribosome. GTP binding is necessary for Tet(O) activity.

Bacterial Proteins↗

PubMed1990

Plant cytosolic ribosomal protein S11 and chloroplast ribosomal protein CS17. Their primary structures and evolutionary relationships.

We have isolated cDNA clones specific for Arabidopsis thaliana cytosolic ribosomal protein S11 and plastid ribosomal protein CS17, both of which are encoded in the nuclear genome, through the use of the corresponding soybean and pea cDNAs as probes, respectively. The nucleotide sequences of all four cDNAs were determined. The amino acid sequences derived from these cDNA sequences show that the soybean and A. thaliana S11 cDNAs encode proteins that are homologous to rat ribosomal protein S11 and that the pea and A. thaliana CS17 cDNAs encode proteins that are homologous to Escherichia coli ribosomal protein S17. The plant S11 cytosolic ribosomal proteins also show significant sequence similarity to both E. coli ribosomal protein S17 and plastid CS17 indicating that these are all related proteins. Comparison of A. thaliana CS17 with A. thaliana S11 and with E. coli S17 suggests that CS17 is more related to S17 than it is to S11. These results support the idea that the gene encoding CS17 was derived from a prokaryotic endosymbiont and not from a duplication of the eukaryotic S11 gene.

Amino Acid Sequence↗

PubMed1977

Ribosomal RNA genes of Saccharomyces cerevisiae. I. Physical map of the repeating unit and location of the regions coding for 5 S, 5.8 S, 18 S, and 25 S ribosomal RNAs.

The organization of the ribosomal DNA repeating unit from Saccharomyces cerevisiae has been analyzed. A cloned ribosomal DNA repeating unit has been mapped with the restriction enzymes Xma 1, Kpn 1, HindIII, Xba 1, Bgl I + II, and EcoRI. The locations of the sequences which code for 5 S, 5.8 S, 18 S, and 25 S ribosomal RNAs have been determined by hybridization of the purified RNA species with restriction endonuclease generated fragments of the repeating unit. The position of the 5.8 S ribosomal DNA sequences within the repeat was also established by sequencing the DNA which codes for 83 nucleotides at the 5' end of 5.8 S ribosomal RNA. The polarity of the 35 S ribosomal RNA precursor has been established by a combination of hybridization analysis and DNA sequence determination and is 5'-18 S, 5.8 S, 25 S-3'.

DNA↗

PubMed1982

Ribosome structure. Localization of 7-methylguanosine in the small subunits of Escherichia coli and chloroplast ribosomes by immunoelectron microscopy.

The minor nucleoside 7-methylguanosine occurs in Escherichia coli 16 S ribosomal RNA at a single site. High pressure liquid chromatographic analysis shows that a single residue of 7-methylguanosine is also present in chloroplast 16 S ribosomal RNA, presumably at an analogous position in the sequence. Antibodies to 7-methylguanosine were induced in rabbits and shown to be highly specific for the intact methylated base. These antibodies were reacted with 30 S ribosomal subunits from E. coli and from the chloroplasts of Alaskan peas. These two types of ribosome have been shown to be topographically similar (Trempe, M. R., and Glitz, D. G. (1981) J. Biol. Chem. 256, 11873-11879). Electron microscopy of the subunit-antibody complexes showed similar subunit-IgG monomers and antibody-linked subunit dimers. In greater than 95% of the complexes observed for each type of ribosome, antibody contact was consistent with a single binding site, which places 7-methylguanosine near the junction of the upper one-third and lower two-thirds of the subunit and maximally distant from the platform. The analogous localization in both E. coli and chloroplast 30 S ribosomal subunits lends support to their proposed common evolutionary origin.

Antigen-Antibody Complex↗

PubMed1980

The effect of antibodies against Escherichia coli small ribosomal subunit proteins on protein synthesis by rat liver ribosomes.

Monovalent Fab fragments from immunoglobulins, directed against Escherichia coli small ribosomal subunit proteins, were tested for their effect on poly(U)-directed synthesis of polyphenylalanine by rat liver ribosomes. Of the 18 Fabs tested, 3, anti-S10, anti-S12, and anti-S14, inhibited polyphenylalanine synthesis; antibodies against 3 of the 21 E. coli ribosomal proteins (S1, S16, and S17) were not tested. The others were either without any appreciable effect or, as in the case of anti-S11 and anti-S18, inhibited far less. Antibodies against S12 and S14 (but not against S10) inhibited elongation factor 1-dependent binding of [14C]Phe-tRNA to rat liver 80 S ribosomes. The presumption is that the prokaryotic (E. coli) ribosomal proteins, S10, S12, and S14, possess antigenic determinants also present in eukaryotic (rat liver) ribosomal proteins; the rat liver proteins have not yet been identified.

Animals↗

PubMed1981

Further characterization of ribosome releasing factor and evidence that it prevents ribosomes from reading through a termination codon.

Ribosome releasing factor, an Escherichia coli protein known to release ribosomes from mRNA at the termination codon, was purified from both the ribosomal wash and the soluble fractions to electrophoretical homogeneity. These preparations had a molecular weight of 23,500 determined by gel electrophoresis, and they were immunologically indistinguishable. Translation of various mRNA was stimulated up to 3-fold upon addition of ribosome releasing factor. On the other hand, amino acid incorporation into proteins programmed by a mutant R17 RNA (an amber mutation at the seventh triplet of coat cistron) was inhibited by this factor. In this system, the major polypeptide formed in the absence of this factor had a molecular weight very close to the authentic R17 coat protein, suggesting that ribosomes may read through the amber codon in the absence of ribosome releasing factor.

Codon↗

PubMed1990

A single base mutation at position 2661 in E. coli 23S ribosomal RNA affects the binding of ternary complex to the ribosome.

A single base substitution mutation from guanine to cytosine was constructed at position 2661 of Escherichia coli 23S rRNA and cloned into the rrnB operon of the multi-copy plasmid pKK3535. The mutant plasmid was transformed into E.coli to determine the effect of the mutation on cell growth as well as the structural and functional properties of the mutant ribosomes in vivo and in vitro. The results show that the mutant ribosomes have a slower elongation rate and an altered affinity for EF-Tu-tRNA-GTP ternary complex. This supports previous findings which indicated that position 2661 is part of a region of 23S rRNA that forms a recognition site for binding of the ternary complex in the ribosomal A site. Combinations of the 2661 mutation with various mutations in ribosomal protein S12 also demonstrate that elements of both ribosomal subunits work in concert to form this binding site.

Cytosine↗

PubMed1989

In vivo selected promoter and ribosome binding site up-mutations: demonstration that the Escherichia coli bla promoter and a Shine-Dalgarno region with low complementarity to the 16 S ribosomal RNA function in Bacillus subtilis.

We have constructed a plasmid, pQS1, in which a mouse dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP:oxidoreductase; EC 1.5.1.3; DHFR) cDNA is inserted in the unique PstI site of a gram-positive/gram-negative shuttle vector derived from pBR322. The cDNA is expressed under the control of the bla promoter, which, like most gram-negative bacterial genes, is considered not to be expressed in Bacillus subtilis, and its coding sequence is translated from a polycistronic message. We have selected in vivo and studied, in Escherichia coli and B. subtilis, expression mutants with promoter and ribosome binding site sequence mutations. One promoter mutation changes the third nucleotide of the -35 region from a C to a G. As expected, this substitution results in increased transcriptional activity in E. coli. In B. subtilis, this mutation induces the accumulation not only of a low but significant amount of dhfr mRNA but also of DHFR, demonstrating that binding strengths with a free energy as low as -9.4 kcal/mol are sufficient to promote ribosome binding in B. subtilis. The association of the promoter mutation (C-G) with a mutation which creates a strong B. subtilis ribosome binding site (-21 kcal/mol) results in the accumulation of a large amount of dhfr mRNA. This demonstrates the importance of having an efficient ribosome binding site in the evaluation of promoter function: for example, with this strong ribosome binding site we can show that the wild-type bla promoter is recognized by the B. subtilis transcription machinery.

Animals↗

PubMed1992

The 5S ribosomal RNA gene is linked to large and small subunit ribosomal RNA genes in the oomycetes, Phytophthora vignae, P. cinnamomi, P. megasperma f.sp. glycinea and Saprolegnia ferax.

Southern hybridization and polymerase chain reaction data indicate that the 5S ribosomal RNA gene is linked to the ribosomal RNA gene repeat unit in the oomycetes, Phytophthora vignae, P. cinnamomi, P. megasperma f.sp. glycinea and Saprolegnia ferax, and is apparently transcribed in the same direction as the large and small subunit ribosomal RNA genes. The polymerase chain reaction has been used to amplify all components of the entire ribosomal RNA gene repeat unit for each of these oomycetes. The total size of all amplified products is identical to the size of the ribosomal RNA gene repeat unit, as determined by Southern analysis.

Base Sequence↗

PubMed2006

Changes in the level of poly(Phe) synthesis in Escherichia coli ribosomes containing mutants of L4 ribosomal protein from Thermus thermophilus can be explained by structural changes in the peptidyltransferase center: a molecular dynamics simulation analysis.

Data from polyphenylalanine [poly(Phe)] synthesis determination in the presence and in the absence of erythromycin have been used in conjunction with Molecular Dynamics Simulation analysis, in order to localize the functional sites affected by mutations of Thermus thermophilus ribosomal protein L4 incorporated in Escherichia coli ribosomes. We observed that alterations in ribosome capability to synthesize poly(Phe) in the absence of erythromycin were mainly correlated to shifts of A2062 and C2612 of 23S rRNA, while in the presence of erythromycin they were correlated to shifts of A2060 and U2584 of 23S rRNA. Our results suggest a means of understanding the role of the extended loop of L4 ribosomal protein in ribosomal peptidyltransferase center.

Amino Acid Sequence↗

PubMed1993

Partial purification of two proteins which sensitize ribosomes to gelonin: sensitization is not linked to phosphorylation of ribosomal proteins.

Inactivation of ribosomes by gelonin, from Gelonium multiflorum, requires ATP and extraribosomal protein(s) present in the rabbit reticulocyte lysate [SPERTI, S. et al. (1991) Biochem. J. 277, 281-284]. On the anion exchanger Mono Q the activity responsible for the sensitization of ribosomes to gelonin resolves in two peaks which both display a kinase activity on ribosomal proteins. However, staurosporin, an inhibitor of several protein kinases, strongly inhibits phosphorylation of ribosomal proteins without affecting the gelonin-promoting activity of Mono Q peaks. All the evidence collected contradicts a direct link between sensitization to gelonin and phosphorylation of ribosomes.

Animals↗

PubMed1987

In vitro synthesis of 16S ribosomal RNA containing single base changes and assembly into a functional 30S ribosome.

Functional 30S ribosomes were reconstructed from total Escherichia coli 30S ribosomal proteins and 16S ribosomal RNA synthesized in vitro by T7 RNA polymerase. Up to 700 mol of RNA/mol of template could be obtained. The transcript lacked all ten normally modified bases and had three additional 5' G residues, an A----G change at position 2, and, in 22% of the molecules, one or two extra 3' residues. The synthetic 16S RNA could be assembled into a particle that cosedimented with authentic 30S and was indistinguishable from 30S by electron microscopy. When supplemented with the 50S subunit, the particles bound tRNA to the 70S P site in a codon- and Mg2+-dependent manner. The specific binding activity was 94% that of particles reconstituted with natural rRNA and 52% that of native 30S. Cross-linking to P site bound tRNA was also preserved. Changing C-1400, the residue known to be close to the anticodon of P site bound tRNA, to A had little effect on reconstitution, but the C----G substitution caused a marked inhibition of assembly. tRNA could bind to both reconstituted mutants, but cross-linking was greatly reduced. These results show that none of the modified bases of 16S RNA are essential for P site binding and that position 1400 may be more important for ribosome assembly than for tRNA binding. Base-specific in vitro mutagenesis can now be used to explore in detail the functional properties of individual residues in ribosomal RNA.

Base Sequence↗

PubMed1999

Directed hydroxyl radical probing of 16S ribosomal RNA in 70S ribosomes from internal positions of the RNA.

Directed hydroxyl radical probing of 16S ribosomal RNA from Fe(II) tethered to specific sites within the RNA was used to determine RNA-RNA proximities in 70S ribosomes. We have transcribed 16S ribosomal RNA in vitro as two separate fragments, covalently attached an Fe(II) probe to a 5'-guanosine-alpha-phosphorothioate at the junction between the two fragments, and reconstituted 30S subunits with the two separate pieces of RNA and the small subunit proteins. Reconstituted 30S subunits capable of association with 50S subunits were selected by isolation of 70S ribosomes. Hydroxyl radicals, generated in situ from the tethered Fe(II), cleaved sites in the 16S rRNA backbone that were close in three-dimensional space to the Fe(II), and a primer extension was used to identify these sites of cleavage. Two sets of 16S ribosomal RNA fragments, 1-360/361-1542 and 1-448/449-1542, were reconstituted into active 30S subunits. Fe(II) tethered to position 361 results in cleavage of 16S rRNA around nucleotides 34, 160, 497, 512, 520, 537, 552, and 615, as well as around positions 1410, 1422, 1480, and 1490. Fe(II) tethered to position 449 induces cleavage around nucleotide 488 and around positions 42 and 617. Fe(II) tethered to the 5' end of 16S rRNA induces cleavage of the rRNA around nucleotides 5, 601, 615, and 642. These results provide constraints for the positioning of these regions of 16S rRNA, for which there has previously been only limited structural information, within the 30S subunit.

Escherichia coli↗

PubMed2004

PRMT3 is a ribosomal protein methyltransferase that affects the cellular levels of ribosomal subunits.

The mammalian protein arginine methyltransferase 3 (PRMT3) catalyzes the formation of asymmetric (type I) dimethylarginine in vitro. As yet, natural substrates and cellular pathways modulated by PRMT3 remain unknown. Here, we have identified an ortholog of PRMT3 in fission yeast. Tandem affinity purification of fission yeast PRMT3 coupled with mass spectrometric protein identification revealed that PRMT3 associates with components of the translational machinery. We identified the 40S ribosomal protein S2 as the first physiological substrate of PRMT3. In addition, a fraction of yeast and human PRMT3 cosedimented with free 40S ribosomal subunits, as determined by sucrose gradient velocity centrifugation. The activity of PRMT3 is not essential since prmt3-disrupted cells are viable. Interestingly, cells lacking PRMT3 showed an accumulation of free 60S ribosomal subunits resulting in an imbalance in the 40S:60S free subunits ratio; yet pre-rRNA processing appeared to occur normally. Our results identify PRMT3 as the first type I ribosomal protein arginine methyltransferase and suggest that it regulates ribosome biosynthesis at a stage beyond pre-rRNA processing.

Amino Acid Motifs↗