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PubMed2000

Interaction of ribosome recycling factor and elongation factor EF-G with E. coli ribosomes studied by the surface plasmon resonance technique.

BACKGROUND: Ribosome recycling factor (RRF), in concert with elongation factor EF-G, is required for disassembly of the post-termination complex of a ribosome after the release of polypeptides. How RRF dissociates the complex has long been puzzling. Crystal structures of RRF molecules have been solved recently and shown to mimic a transfer RNA (tRNA) shape, which prompted us to examine whether RRF binds to the ribosome as tRNA does. RESULTS: The formation of ribosome complexes on the surface-coupled RRF and elongation factor EF-G of Escherichia coli was monitored in real time with a BIACORE 2000 instrument based on the surface plasmon resonance technique. RRF interacted with 70S ribosomes as well as 50S and 30S subunits, although it interacted preferentially with 50S subunits, which was clearly seen under high but physiological ionic conditions. This 50S interaction was diminished by a single amino acid substitutions for Arg132 of RRF, which did not appreciably affect the protein folding but nullified the activity in vivo and in vitro. Moreover, a set of antibiotics that inhibited the RRF-50S interaction were also inhibitory to the polysome breakdown activity of RRF in vitro. The BIACORE technique also worked very well in demonstrating the action of the antibiotics thiostrepton and fusidic acid, which are inhibitory to the RRF function by freezing the pre- and post-translocation intermediates catalysed by EF-G. CONCLUSIONS: These results suggest that the preferential interplay of RRF with the 50S subunit may be of biological significance, probably reflecting the mode of RRF action. The BIACORE technique proved useful for real-time monitoring of the interaction between the ribosome and translation factors, as well as for screening of potential inhibitors for ribosome recycling factor.

Amino Acid Substitution↗
PubMed2001

The large subunit of the mammalian mitochondrial ribosome. Analysis of the complement of ribosomal proteins present.

Identification of all the protein components of the large subunit (39 S) of the mammalian mitochondrial ribosome has been achieved by carrying out proteolytic digestions of whole 39 S subunits followed by analysis of the resultant peptides by liquid chromatography and mass spectrometry. Peptide sequence information was used to search the human EST data bases and complete coding sequences were assembled. The human mitochondrial 39 S subunit has 48 distinct proteins. Twenty eight of these are homologs of the Escherichia coli 50 S ribosomal proteins L1, L2, L3, L4, L7/L12, L9, L10, L11, L13, L14, L15, L16, L17, L18, L19, L20, L21, L22, L23, L24, L27, L28, L30, L32, L33, L34, L35, and L36. Almost all of these proteins have homologs in Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae mitochondrial ribosomes. No mitochondrial homologs to prokaryotic ribosomal proteins L5, L6, L25, L29, and L31 could be found either in the peptides obtained or by analysis of the available data bases. The remaining 20 proteins present in the 39 S subunits are specific to mitochondrial ribosomes. Proteins in this group have no apparent homologs in bacterial, chloroplast, archaebacterial, or cytosolic ribosomes. All but two of the proteins has a clear homolog in D. melanogaster while all can be found in the genome of C. elegans. Ten of the 20 mitochondrial specific 39 S proteins have homologs in S. cerevisiae. Homologs of 2 of these new classes of ribosomal proteins could be identified in the Arabidopsis thaliana genome.

Amino Acid Sequence↗
PubMed1989

The gene and the primary structure of acidic ribosomal protein A0 from yeast Saccharomyces cerevisiae which shows partial homology to bacterial ribosomal protein L10.

Eukaryotic ribosomes contain an acidic ribosomal protein of about 38 kDa which shows immunological cross-reactivity with the 13 kDa-type acidic ribosomal proteins that are related to L7/L12 of bacterial ribosomes. By using a cDNA clone for 38 kDa-type acidic ribosomal protein A0 from the yeast Saccharomyces cerevisiae, we have cloned a genomic DNA encoding A0 and determined the sequence of 1,614 nucleotides including about 500 nucleotides in the 5'-flanking region. The gene lacks introns and possesses two boxes homologous to upstream activation sequences (UASrpg) in the 5'-flanking region. The amino acid sequence of A0 deduced from the nucleotide sequence shows that A0 shares a highly similar carboxyl-terminal region of about 40 amino acids in length with 13 kDa-type acidic ribosomal proteins, including an identical carboxyl-terminal, DDDMGFGLFD. In the amino-terminal region A0 contains an arginine-rich segment which shows a low but distinct similarity to that of bacterial ribosomal protein L10 through which L10 is thought to bind to 23S rRNA. On the other hand, the carboxyl-terminal half of A0 is enriched with hydrophobic amino acid residues including four pairs of phenylalanine residues which are all conserved in a human homologue.

Amino Acid Sequence↗
PubMed2004

Silencing of ribosomal protein L3 genes in N. tabacum reveals coordinate expression and significant alterations in plant growth, development and ribosome biogenesis.

The expression of ribosomal protein genes is coordinately regulated in bacteria, yeast, and vertebrates, so that equimolar amounts of ribosomal proteins accumulate for assembly into ribosomes. To understand how expression of ribosomal protein genes is regulated in plants, we altered expression of the large subunit ribosomal protein L3 (RPL3) genes in Nicotiana tabacum using post-transcriptional gene silencing (PTGS). L3 is encoded by two genes, RPL3A and RPL3B, with 80.2% amino acid sequence identity in tobacco. Two types of 'hairpin' RNA (hpRNA) vectors carrying the RPL3A or RPL3B sequences in both sense and antisense orientation were generated in order to alter the expression level of both RPL3 genes. Tobacco plants transformed with a vector containing a 5'-terminal fragment of RPL3A gene displayed decreased RPL3A mRNA levels and a marked increase in the abundance of RPL3B mRNA. These results indicated that expression of the RPL3 genes is coordinately regulated in tobacco. The transgenic plants that contained higher levels of RPL3B mRNA exhibited leaf overgrowth and mottling. Epidermal cells of these plants were increased in number and decreased in size. The precursor rRNA (pre-rRNA) and the mature rRNAs accumulated in these plants, suggesting that ribosome biogenesis is upregulated. Tobacco plants transformed with an hpRNA vector harboring the full-length RPL3B cDNA exhibited efficient silencing of both RPL3A and RPL3B genes, reduced L3 levels, and an abnormal phenotype characterized by a delay in development, stunting, and inhibition of lateral root growth. L3 deficiency led to a reduction in cell number and an increase in cell size, suggesting that L3 positively regulates cell division. Decreasing RPL3 gene expression resulted in a decrease in accumulation of the pre-rRNA, establishing a prominent role for L3 in ribosome biogenesis in plants.

Gene Expression Regulation, Plant↗
PubMed2005

Translational operator of mRNA on the ribosome: how repressor proteins exclude ribosome binding.

The ribosome of Thermus thermophilus was cocrystallized with initiator transfer RNA (tRNA) and a structured messenger RNA (mRNA) carrying a translational operator. The path of the mRNA was defined at 5.5 angstroms resolution by comparing it with either the crystal structure of the same ribosomal complex lacking mRNA or with an unstructured mRNA. A precise ribosomal environment positions the operator stem-loop structure perpendicular to the surface of the ribosome on the platform of the 30S subunit. The binding of the operator and of the initiator tRNA occurs on the ribosome with an unoccupied tRNA exit site, which is expected for an initiation complex. The positioning of the regulatory domain of the operator relative to the ribosome elucidates the molecular mechanism by which the bound repressor switches off translation. Our data suggest a general way in which mRNA control elements must be placed on the ribosome to perform their regulatory task.

Bacterial Proteins↗
PubMed2005

Rrp15p, a novel component of pre-ribosomal particles required for 60S ribosome subunit maturation.

In eukaryotes ribosome biogenesis required that rRNAs primary transcripts are assembled in pre-ribosomal particles and processed. Protein factors and pre-ribosomal complexes involved in this complex pathway are not completely depicted. The essential ORF YPR143W encodes in yeast for an uncharacterized protein product, named here Rrp15p. Cellular function of Rrp15p has not so far defined even if nucleolar location was referred. With the aim to define the possible role of this orphan gene, we performed TAP-tagging of Rrp15p and investigated its molecular association with known pre-ribosomal complexes. Comparative sucrose gradient sedimentation analyses of yeast lysates expressing the TAP-tagged Rrp15p, strongly indicated that this protein is a component of the pre-60S particles. Northern hybridization, primer extension and functional proteomics on TAP-affinity isolated complexes proved that Rrp15p predominately associated with pre-rRNAs and proteins previously characterized as components of early pre-60S ribosomal particles. Finally, depletion of Rrp15p inhibited the accumulation of 27S and 7S pre-rRNAs and 5.8S and 25S mature rRNA. These results provide the first indication that Rrp15p is a novel factor involved in the early maturation steps of the 60S subunits. Moreover, the identification of the protein kinase CK2 in the Rrp15p-containing pre-ribosomal particles here reported, sustains the link between ribosome synthesis and cell cycle progression.

Amino Acid Sequence↗
PubMed1986

Construction and functional analysis of ribosomal 5S RNA from Escherichia coli with single base changes in the ribosomal protein binding sites.

The ribosomal 5S RNA gene from E. coli was altered by oligonucleotide-directed mutagenesis at positions A66 and U103. The mutant genes were cloned into an expression vector and selectively transcribed in an UV-sensitive E. coli strain using a modified maxicell system. The mutant 5S RNA genes were found to be transcribed and processed normally. The 5S RNA molecules were assembled into 50S ribosomal subunits. Under in vitro conditions the stability of the mutant 70S ribosomes seemed, however, to be reduced, since they dissociated into their subunits more easily than those of the wild type. The isolated mutated 5S RNAs with base changes in the ribosomal protein binding sites for L18 and L25, together with a point mutant at G41 (G to C), constructed earlier, were tested for their capacity to bind the 5S RNA binding proteins L5, L18 and L25. The following effects were observed: The base change A66 to C within the L18 binding site did not affect the binding of the ribosomal protein L18 but enhanced the stability of the L25-5S RNA complex considerably. The base changes U103 to G and G41 to C slightly reduced the binding of L5 and L25 whereas the binding of L18 to the mutant 5S RNAs was not altered. In addition 70S ribosomes with the single point mutations in their 5S RNAs were tested in their tRNA binding capacity. Mutants containing a C41 in their 5S RNA showed a reduction in the poly(U)-dependent Phe-tRNA binding, whereas the mutations to C66 and G 103 lead to completely inactive ribosomes in the same assay. Based on previous results a spatial model of the 5S RNA molecule is presented which is consistent with the findings reported in this paper.

Cloning, Molecular↗
PubMed1981

Ribosomal proteins S9 and L6 participate in the binding of [3H]dibekacin to E. coli ribosomes.

The degrees of binding of [3H]dibekacin to LiCl-treated cores of E. coli ribosomes were reduced by increasing LiCl concentrations. The 1.15 M LiCl core lost 70 approximately 80% of the original binding capacity. The antibiotic attachment to the 1.15 M LiCl core was restored by reconstitution with the split proteins (SP), which were obtained by the treatment of 70S ribosomes with LiCl at concentrations of 0.8 approximately 1.15 M. The basic proteins, split off during the transition from 0.4 M LiCl core to 0.8 approximately 1.15 M LiCl core, seemed to be involved in the drug binding. SP0.4 approximately 1.15, which was obtained by the treatment of the 0.4 M LiCl core with 1.15 M LiCl, was fractionated by CM-Sephadex C-25 column chromatography, and each fraction was assayed for protein composition and the capability of restoring the ability of the 1.15 M LiCl core to bind the drug. Of ribosomal proteins eliminated with 1.15 M LiCl, the addition of either S9 or L6 alone to the 1.15 M LiCl core was observed to restore approximately 50% of the binding as compared to the 70S ribosome alone, and both proteins restored about 70% of the binding. The results suggested that ribosomal proteins S9 and L6 were involved in the attachment of [3H]dibekacin to the ribosome. The antibiotic binding to the 70S ribosome and 1.15 M LiCl core reconstituted with S9 and L6 was considerably inhibited by unlabelled dibekacin or kanamycin, and partially inhibited by gentamicin or neomycin, but was not significantly affected by streptomycin or viomycin.

Aminoglycosides↗
PubMed1994

Localization of Saccharomyces cerevisiae ribosomal protein L16 on the surface of 60 S ribosomal subunits by immunoelectron microscopy.

Antibodies raised against a trpE-L16 fusion protein expressed in Escherichia coli were used to examine immunological relatedness between Saccharomyces cerevisiae ribosomal protein L16 and ribosomal proteins from eubacteria, halobacteria, methanogens, eocytes, and other eukaryotes. Homologues of L16 also were identified by searches of sequence data bases. Among the bacterial proteins that are immunologically related and similar in sequence to L16 are ribosomal proteins that bind 5 S rRNA. L16 protein fused near its carboxyl terminus to E. coli beta-galactosidase could assemble into functional yeast 60 S ribosomal subunits. The RPL16A-lacZ gene fusion partially complemented the slow growth or lethality of mutants containing null alleles of one or both RPL16 genes, respectively. L16-beta-galactosidase fusion protein cosedimented with ribosomes and polyribosomes, and remained associated with high salt-washed ribosomes. Monoclonal antibodies against beta-galactosidase were used to map the location of L16-beta-galactosidase on the surface of the 60 S subunit by immunoelectron microscopy. L16 was localized near the top surface of the central protuberance, where the 60 S subunit potentially contacts the 40 S subunit. This is similar to the location of the bacterial homologues of L16 in 50 S ribosomal subunits.

Amino Acid Sequence↗
PubMed1997

Protein kinase associated with ribosomes phosphorylates ribosomal proteins of Streptomyces collinus.

Protein kinase activity associated with ribosomes of a kirromycin-producing strain of Streptomyces collinus was detected. The enzyme utilizes [gamma-32P]ATP to phosphorylate proteins, yielding acid-stable phosphoamino acids. Two-dimensional electrophoresis of proteins from a crude ribosomal fraction revealed 17 phosphoproteins. Eleven of the phosphoproteins exhibited electrophoretic mobility identical to that of S. collinus ribosomal proteins S3, S4, S12, S13, S14, S18, L2, L7, L16, L17, and L23. Protein L2 was identified by microsequencing of internal peptide fragments. Immunodetection with monoclonal antibodies indicated that the ribosomal proteins are phosphorylated on serine and threonine residues. Phosphorylation of ribosomal proteins led to the reduction of activity of ribosomes in the translation of poly(U). These results provide the first evidence of phosphorylation of ribosomal proteins in bacteriophage-uninfected cells of eubacteria.

Bacterial Proteins↗
PubMed1988

Chloroplast ribosomal protein L-18 in Chlamydomonas reinhardtii is processed during ribosome assembly.

Chloroplast ribosomal protein L-18 is made in the cytoplasm as a precursor, imported into the chloroplast, and processed to the mature form in two steps. We report here that the intermediate produced following the first processing step associates specifically with a ribosomal complex migrating with the chloroplast ribosome large subunit peak in sucrose gradients, and is then processed into mature L-18. This processing event is slowed down in mutant cells deficient in synthesis of non-ribosomal proteins in the chloroplast. Thus the second processing step of L-18 occurs during ribosome assembly, depends on one or more nonribosomal proteins made in the chloroplast, and may be required for the maturation of the 50 S ribosome subunit. The mature L-18 protein shows extensive sequence homology at its amino-terminus to Escherichia coli ribosomal protein L27, which is located at the interface between 30 S and 50 S subunits and is involved in the formation of the peptidyl-tRNA binding site.

Amino Acid Sequence↗
PubMed1984

Towards a classification of E. coli ribosomal proteins: a hypothetical 'small ribosome' as a primitive protein-synthesizing apparatus.

Homologies were searched among the published primary sequences of 51 E. coli ribosomal proteins, partly by 'eye' and partly by computer-assisted methods. By employing Moore and Goodman's alignment statistics for evaluating homology levels, 33 out of these 51 ribosomal proteins has been classified into 9 homology groups, some of which being yet tentative and remaining to be further analyzed. Taking it into consideration that most ribosomal protein genes are clustered at str-stc region, rif region and several other regions, these results strongly suggest that most or all of the contemporary ribosomal proteins must have evolved by repeated gene duplications of very few (or only one) primitive ancestral ribosomal protein gene(s). Thus it is most reasonable to propose that 'a small ribosome' consisting of very few (or only one) ribosomal protein(s) must have existed as a primitive protein-synthesizing apparatus.

Amino Acid Sequence↗
PubMed2004

Ribosome modulation factor protects Escherichia coli during heat stress, but this may not be dependent on ribosome dimerisation.

The role of ribosome modulation factor (RMF) in protecting heat-stressed Escherichia coli cells was identified by the observation that cultures of a mutant strain lacking functional RMF (HMY15) were highly heat sensitive in stationary phase compared to those of the parent strain (W3110). No difference in heat sensitivity was observed between these strains in exponential phase, during which RMF is not synthesised. Studies by differential scanning calorimetry demonstrated that the ribosomes of stationary-phase cultures of the mutant strain had lower thermal stability than those of the parent strain in stationary phase, or exponential-phase ribosomes. More rapid breakdown of ribosomes in the mutant strain during heating was confirmed by rRNA analysis and sucrose density gradient centrifugation. Analyses of ribosome composition showed that the 100S dimers dissociated more rapidly during heating than 70S particles. While ribosome dimerisation is a consequence of the conformational changes caused by RMF binding, it may not therefore be essential for RMF-mediated ribosome stabilisation.

Binding Sites↗
PubMed1977

On the control of ribosomal protein biosynthesis in Escherichia coli. I. Studies on ribosomal protein biosynthesis in amino acid-starved cells.

The rate of individual ribosomal protein synthesis relative to total protein synthesis has been determined in Escherichia coli rel+ and rel- cells, under valyltRNA deprivation. These strains have a temperature-sensitive valyl-tRNA synthetase. Starvation was obtained following transfer to the cells to non-permissive temperature. Ribosomal proteins were obtained by treatment of either total lysates of freeze-thawed lysozyme spheroplasts or ammonium sulphate precipitate of ribosomes, with acetic acid. Differential labelling of the ribosomal proteins was observed in both strains: proteins from the rel+ strain appear more labelled than those from the rel- strain, the rate of labelling of individual proteins being about the same in both strains. Moreover ribosomal proteins were found as stable during starvation as total protein. It is thus concluded that in starving cells individual ribosomal proteins are not synthesized at equal rates. This indicates that the synthesis of ribosomal proteins is not only under the control of the rel gene.

Amino Acids↗
PubMed1979

Ribosomal proteins synthesis and exchange in the absence of 28-S and 18-S ribosomal RNA synthesis in L5178Y cells.

The effect of the adenosine analogue toyocamycin on ribosomal proteins synthesis and assembly within ribosomal particles was investigated in the murine cells, L5178Y. The analogue was used for periods not exceeding 5 h, at a concentration which permits the synthesis of ribosomal precursor RNA but inhibits the maturation process. The following observations were made: 1. Ribosomal proteins, synthesized de novo in the presence of the drug, were associated with toyocamycin-containing 45-S pre-rRNA in preribosomal-like 80-S ribonucleoproteins which accumulated in the nucleolus. Two-dimensional electrophoresis revealed a full protein complement of these particles, although minor discrepancies were observed in the relative proportions of a limited number of polypeptides. 2. In the absence of 28-S and 18-S rRNA formation, a surprisingly high proportion of newly synthesized ribosomal proteins were incorporated into high-salt washed ribosomal subunits. The extent of individual protein exchange as well as their apparent turnover rates were markedly heterogeneous. Most of these exchangeable proteins were shown to be labeled rapidly in ribosomal subunits of normal cells. Some alternative interpretations of these results are discussed.

Animals↗
PubMed1981

Protection of rat liver 80 S ribosomes against ricin A chain inactivation by proteins extracted from rat liver and wheat germ ribosomal subunits with ammonium chloride/magnesium chloride.

Proteins extracted from wheat germ 60 S ribosomal subunits and rat liver 60 S and 40 S ribosomal subunits with 3 M NH4Cl/75 mM MgCl2 were able to prevent the ricin A chain-mediated inactivation of untreated 80 S rat liver ribosomes. The protection of polyphenylalanine synthetic capability of 80 S ribosomes was saturable and reached 100% protection in the presence of about 20 micrograms of extracted protein using a uniform set of assay conditions. No protection was observed using proteins extracted from wheat germ 40 S subunits or the core fraction of rat liver 60 S subunits or protein extracted from Escherichia coli ribosomes or ribosomal subunits. The conclusion that the protective effect of extracted 60 S subunit proteins was specific, was further strengthened by showing that unrelated proteins such as alpha-lactalbumin, bovine serum albumin and lysozyme, and polypeptides such as polylysine and poly(aspartic acid), also showed no protection. If 80 S ribosomes were first treated with ricin A chain and then incubated with proteins extracted from rat liver 60 S subunits, no protection was observed. Proteins extracted with NH4Cl/MgCl2 from 60 S rat liver subunits were applied to carboxymethylcellulose column equilibrated with 6 M urea. Stepwise elution with increasing concentrations of LiCl resulted in seven fractions. One fraction (D) contained most of the protective factor; one fraction (E) contained a lesser amount of the protective factor. Two-dimensional polyacrylamide gel electrophoresis of fraction D showed the presence of ten proteins. These data are consistent with the idea that the enzymatic target of ricin A chain is protein is nature and that fraction D contains one or more proteins that appear to act as a inhibitor against ricin A chain.

Ammonium Chloride↗
PubMed1981

Differences in the distribution of phosphate content in the ribosomal subunit proteins of free and membrane-bound ribosomes from normal and regenerating rat liver.

Proteins of membrane-bound ribosomes from normal liver contained 60-70% more phosphate than did proteins from free ribosomes. This difference was not a reflection of the phosphate contents of respective 40 S subunits. Instead, it was owing to the presence of high levels of phosphorylated proteins in the 60 S subunits, i.e., phosphate contents equal to or greater than those for 40 S subunits. The proteins of membrane-bound 60 S subunits contained twice the phosphate as free 60 S subunits. In regenerating rat liver, membrane-bound ribosomes had increased phosphate in the proteins of the 40 S subunits and decreased phosphate in proteins of the 60 S subunit when compared to controls for normal rat liver. No significant changes occurred in the proteins of free ribosomes from regenerating rat liver. These findings are discussed with respect to (a) the importance of assessing total phosphate contents of proteins in the study of ribosomal protein phosphorylation, and (b) the possible involvement of ribosomal protein phosphorylation in the segregation of ribosomes into free and membrane-bound populations and the regulation of these distributions to meet changes in the translational demands of the cell.

Animals↗
PubMed1986

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.

Animals↗