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At least 685 records · Page 38Linked to original sources
PubMed2006

Molecular localization of a ribosome-dependent ATPase on Escherichia coli ribosomes.

We have previously isolated and described an Escherichia coli ribosome-bound ATPase, RbbA, that is required for protein synthesis in the presence of ATP, GTP and the elongation factors, EF-Tu and EF-G. The gene encoding RbbA, yhih, has been cloned and the deduced protein sequence harbors two ATP-motifs and one RNA-binding motif and is homologous to the fungal EF-3. Here, we describe the isolation and assay of a truncated form of the RbbA protein that is stable to overproduction and purification. Chemical protection results show that the truncated RbbA specifically protects nucleotide A937 on the 30S subunit of ribosomes, and the protected site occurs at the E-site where the tRNA is ejected upon A-site occupation. Other weakly protected bases in the region occur at or near the mRNA binding site. Using radiolabeled tRNAs, we study the stimulating effect of this truncated RbbA on the binding and release of different tRNAs bound to the (aminoacyl) A-, (peptidyl) P- and (exit) E-sites of 70S ribosomes. The combined data suggest plausible mechanisms for the function of RbbA in translation.

Adenosine Triphosphatases↗
PubMed1979

Proteins of small subunits of rat liver ribosomes that interact with poly(U). II. Cross-links between poly(U) and ribosomal proteins in 40 S subunits induced by UV irradiation.

(1) When rat liver 40 S ribosomal proteins in 6 M urea were were mixed with poly(U) at an appropriate ratio, a precipitate was formed which was also insoluble in the sample solution for two-dimensional acrylamide gel electrophoresis. Analyses by two-dimensional acrylamide gel electrophoresis showed that S7 and S10 proteins (according to our numbering system) had disappeared selectively from the fraction soluble in 6 M urea. These two proteins were present in the fraction insoluble in 6 M urea, and became soluble in the sample solution after treating it with RNase. The results suggest that S7 and S10 proteins have strong affinities for poly(U). When rat liver 40 S subunits were incubated with poly(U), similar results were obtained. (2) After incubation of 40 S subunits with [3H]poly(U) and then with unlabeled poly(U), UV irradiation cross-linked poly(U) to the protein moiety of the 40 S subunit. When the protein fraction insoluble in the sample solution for two-dimensional electrophoresis was prepared from 40 S subunits cross-linked to poly(U) and then subjected to two-dimensional acrylamide gel electrophoresis after RNase treatment, S7 and S10 proteins were detected on the gel. In addition to the S7 protein spot, a triangular area spreading from the spot to the origin contained radioactivity. The results suggest that poly(U) is cross-linked to S7 protein and oligo(U) fragments bound to S7 protein affect its electrophoretic mobility. (3) Ribosomal proteins were prepared from 40 S subunits cross-linked to carrier-free [3H]poly(U) and analyzed by three-dimensional acrylamide gel electrophoresis (Terao, K. & Ogata, K. (1975) Biochim. Biophys. Acta 402, 214--229) after RNase treatment. It was found that S7, S6, and S15 proteins are cross-linked to poly(U). From the results of the present and preceding experiments it is concluded that S7 is the poly(U)-binding protein. The possibility that other proteins in 40 S ribosomal subunits interact with poly(U) is discussed.

Animals↗
PubMed1993

Solution of the ribosome riddle: how the ribosome selects the correct aminoacyl-tRNA out of 41 similar contestants.

Three tRNA binding sites, the A, P and E sites, have been demonstrated on ribosomes of bacterial, archaebacterial and eukaryotic origin. In all these cases the first and the third site, the A and the E site, are allosterically coupled in the sense of a negative co-operativity. Therefore, the allosteric three-site model seems to be a generally valid description of the ribosomal elongation phase, where in a cycle of reactions the nascent peptide chain is prolonged by one amino acid. The molecular concept of the allosteric three-site model explains the astonishing ability of the ribosome to select the correct substrate out of a large number of very similar substrates, and it provides a framework within which the mechanisms of the elongation factors could be understood.

Allosteric Regulation↗
PubMed2005

DksA represses ribosomal gene transcription in Pseudomonas aeruginosa by interacting with RNA polymerase on ribosomal promoters.

In Escherichia coli transcription of ribosomal RNA (rRNA) is regulated by the H-NS and Fis proteins, as well as by the small signal molecule ppGpp and the initiating nucleotides. During amino acid starvation, the concentration of ppGpp increases, and binding of this alarmone to RNA polymerase (RNAP) leads to inhibition of rRNA transcription, a regulatory event called stringent response. Here we show that in Pseudomonas aeruginosa DksA, a protein with pleiotropic effects, is a negative regulator of rRNA transcription both during exponential growth and stringent conditions. A dksA mutant overexpresses rRNA, without being affected in the production of ppGpp. Cell-fractionation and chromosome immunoprecipitation experiments demonstrate that DksA is associated with DNA, in particular with promoters of ribosomal genes in vivo. The binding to rRNA promoters specifically increases during stringent response, and correlates with the binding of RNAP to these regions. Moreover DksA can be copurified with RNAP subunits in vivo. DNA band shift experiments show that DksA, in synergy with ppGpp, increases the binding of RNAP to ribosomal promoters. Therefore DksA might be a new regulator of rRNA transcription in P. aeruginosa.

Bacterial Proteins↗
PubMed2006

Increased expression of ribosome recycling factor is responsible for the enhanced protein synthesis during the late growth phase in an antibiotic-overproducing Streptomyces coelicolor ribosomal rpsL mutant.

K88E mutation within rpsL, which encodes the S12 ribosomal protein, enhanced the protein synthetic activity of Streptomyces coelicolor during the late growth phase, resulting in overproduction of the deep blue-pigmented polyketide antibiotic actinorhodin. In vitro cross-mixing experiments using the ribosomal and S-150 fractions derived from wild-type and K88E mutant strains suggested that one or more translation factors are enriched in the mutant's S-150 fraction, while Western analysis using antibodies against various translation factors revealed ribosome recycling factor (RRF) to be one of the enriched mediators. RRF purified from overexpressing cells stimulated mRNA-directed green fluorescent protein (GFP) synthesis in an in vitro protein synthesis system. GFP synthesis rates were complemented by RRF addition into wild-type cell's S-150 fraction, eliminating the difference between wild-type and mutant S-150 fractions. The frr gene encoding RRF was found to be transcribed from two distinct start points (frrp1 and frrp2), and increased expression from frrp1 could account for the elevated level of RRF in the K88E mutant during the late growth phase. Moreover, introduction of a plasmid harbouring a high copy number of frr gene into wild-type S. coelicolor induced remarkable overproduction of antibiotic, demonstrating that the increased levels of RRF caused by the K88E mutation is responsible for an aberrant stationary-phase event: overproduction of antibiotic.

Anthraquinones↗
PubMed1985

Molecular cloning and analysis of cDNA sequences for two ribosomal proteins from Artemia. The coordinate expression of genes for ribosomal proteins and elongation factor 1 during embryogenesis of Artemia.

The large subunit of eukaryotic ribosomes contains acidic phosphoproteins which are related to L7/L12 from Escherichia coli. In the brine shrimp Artemia these proteins are designated eL12 and eL12'. We have isolated cDNA clones for these proteins from a cDNA bank that was constructed by the use of size-fractionated poly(A)-rich RNA (8-10S fraction) from Artemia and a synthetic oligonucleotide as primer. Clones containing DNA sequences coding for eL12 and eL12 were characterized by hybrid-selected translation and DNA sequencing. The proteins eL12 and eL12' share an identical peptide of 22 amino acids at their carboxy termini whereas the remaining part of the protein shows little sequence homology. The nucleotide sequences show a different codon use for the amino acids in the common carboxy terminus, thereby excluding a common exon coding for this part of both proteins. Despite the differences in amino acid sequence in the major part of eL12 and eL12' the proteins have a considerable degree of homology on the basis of the distribution of hydrophobic and hydrophilic amino acids over the polypeptide chains, in agreement with a related folding and function of both proteins. Relative levels of mRNA coding for eL12, eL12' and elongation factor 1 alpha were determined during the development of Artemia from a dormant cyst to a nauplius. The data show a coordinate expression of the genes for EF-1 alpha and both ribosomal proteins, excluding a differential expression of the genes for these related ribosomal proteins during embryogenesis. Analysis of the gene copy number for eL12 and eL12' indicates the presence of a few genes for each protein.

Animals↗
PubMed1985

A mutant from Escherichia coli which lacks ribosomal proteins S17 and L29 used to localize these two proteins on the ribosomal surface.

A mutant of Escherichia coli has been isolated which lacked ribosomal proteins S17 and L29, as judged by two-dimensional gel electrophoresis. A battery of immunological tests was used to confirm this result. Ribosomes of this mutant were used as a control for the localization of proteins S17 and L29 on the surface of the ribosomal subunits of E. coli. Protein S17 has been localized on the 30S subunit body, 3-5 nm away from the lower pole, while protein L29 is located at the back of the 50S particle on the opposite side to the interface.

Bacterial Proteins↗
PubMed1989

Primary structures of five ribosomal proteins from the archaebacterium Halobacterium marismortui and their structural relationships to eubacterial and eukaryotic ribosomal proteins.

The complete amino acid sequences of ribosomal proteins L9, L20, L21/22, L24 and L32 from the archaebacterium Halobacterium marismortui were determined. The comparison of the sequences of these proteins with those from other organisms revealed that proteins L21/22 and L24 are homologous to ribosomal protein Yrp29 from yeast and L19 from rat, respectively, and that H. marismortui L20 is homologous to L30 from eubacteria. H. marismortui ribosomal protein L9 showed sequence homology to both L29 from yeast and L15 from eubacteria. No homologous protein was found for H. marismortui L32. These results are discussed with respect to the phylogenetic relationship between eubacteria, archaebacteria and eukaryotes.

Amino Acid Sequence↗
PubMed1992

Purification, primary structure and molecular cloning of a rat ribosomal protein showing homology with yeast ribosomal protein YL34.

1. A new 17-kDa mammalian ribosomal protein (PR17) was purified to homogeneity from the rat exocrine pancreas. The purification procedure was based on acidic extraction of a heat-denatured homogenate, ammonium-sulfate precipitation, hydrophobic chromatography on phenyl-Sepharose and analytical reverse-phase HPLC on mu Bondapak C18. Fractions of interest were collected using an antiserum directed against the first (1-14) moiety of somatostatin (1-28). 30 micrograms pure RP17 were obtained from 1 g fresh pancreas. 2. A short 111-b cDNA encoding RP17 was amplified from rat pancreatic first-strand cDNA template by using two 64-fold degenerate heptadecamer primers in the DNA-polymerase-chain reaction. From the sequence of amplified cDNA, an unambiguous oligonucleotide probe was designed to screen a rat pancreatic cDNA library. A cDNA clone coding for RP17 was isolated, whose nucleotide sequence, with an open reading frame coding for 155 amino acids (molecular mass of 17,199 Da), confirmed the partial amino acid sequences directly obtained from the purified protein. 3. Northern-blot analysis showed that a similar 0.75-kb transcript was present in rat pancreas, in the rat pancreatic acinar cell line AR 4-2J and in the human neuroblastoma cell-line NB-OK-1, the highest level being in the latter two preparations, despite similar levels of RP17 in all three preparations, as tested with a rabbit antiserum directed against purified RP17. 4. The N-terminal sequence of both RP17 and the ribosomal protein YL43 from Saccharomyces cerevisiae (39 amino acid residues) showed a high degree of identity (77%), indicating that RP17 is a mammalian homolog of yeast ribosomal protein YL43.

Amino Acid Sequence↗
PubMed1975

Antibacterial action of primaquine: effects in vitro on polypeptide synthesis and in vivo on ribosomes and ribosomal ribonucleic acid.

Primaquine inhibited polyphenylalanine formation directed by poly(U) in a cell-free system obtained from Bacillus megaterium only when the drug was preincubated with transfer ribonucleic acid (tRNA), poly(U), or ribosomes. Considerably less inhibition was produced when the ionic strength of the preincubation mixture of tRNA or poly(U) plus primaquine was increased; with ribosomes, the extent of inhibition was only slightly reduced. In cultures of B. megaterium, primaquine induced the breakdown of ribosomes and their RNA.

Bacillus megaterium↗
PubMed1974

Amino-terminal sequences of some Escherichia coli 30S ribosomal proteins and functionally corresponding Bacillus stearothermophilus ribosomal proteins.

Amino-terminal sequences of five purified Escherichia coli 30S ribosomal proteins (S4, S9, S10, S16, and S20) were compared with those of their functionally corresponding Bacillus stearothermophilus ribosomal proteins identified previously by the reconstitution technique. An automatic Edman degradation method was used for sequence determinations. The sequence of the first 30 residues is presented, except that only the first 25 residues are shown for the S20 pair. Substantial (40 to 70%) sequence homologies have been observed in every case. The results show that the pairs of functionally equivalent proteins, previously identified by the reconstitution technique, are also chemically related. Thus, the present chemical studies give further support for the previous conclusion that two ribosomes with different properties, 30S subunits from E. coli and B. stearothermophilus, have the same fundamental structural organization.

Amino Acid Sequence↗
PubMed1981

Regulation of ribosomal protein synthesis in an Escherichia coli mutant missing ribosomal protein L1.

In an Escherichia coli B strain missing ribosomal protein L1, the synthesis rate of L11 is 50% greater than that of other ribosomal proteins. This finding is in agreement with the previous conclusion that L1 regulates synthesis of itself and L11 and indicates that this regulation is important for maintaining the balanced synthesis of ribosomal proteins under physiological conditions.

Bacterial Proteins↗
PubMed2002

Leaderless mRNAs bind 70S ribosomes more strongly than 30S ribosomal subunits in Escherichia coli.

By primer extension inhibition assays, 70S ribosomes bound with higher affinity, or stability, than did 30S subunits to leaderless mRNAs containing AUG or GUG start codons. Addition of translation initiation factors affected ribosome binding to leaderless mRNAs. Our results suggest that translation of leaderless mRNAs might initiate through a pathway involving 70S ribosomes or 30S subunits lacking IF3.

Codon, Initiator↗
PubMed2005

A peptide derived from RNA recognition motif 2 of human la protein binds to hepatitis C virus internal ribosome entry site, prevents ribosomal assembly, and inhibits internal initiation of translation.

Human La protein is known to interact with hepatitis C virus (HCV) internal ribosome entry site (IRES) and stimulate translation. Previously, we demonstrated that mutations within HCV SL IV lead to reduced binding to La-RNA recognition motif 2 (RRM2) and drastically affect HCV IRES-mediated translation. Also, the binding of La protein to SL IV of HCV IRES was shown to impart conformational alterations within the RNA so as to facilitate the formation of functional initiation complex. Here, we report that a synthetic peptide, LaR2C, derived from the C terminus of La-RRM2 competes with the binding of cellular La protein to the HCV IRES and acts as a dominant negative inhibitor of internal initiation of translation of HCV RNA. The peptide binds to the HCV IRES and inhibits the functional initiation complex formation. An Huh7 cell line constitutively expressing a bicistronic RNA in which both cap-dependent and HCV IRES-mediated translation can be easily assayed has been developed. The addition of purified TAT-LaR2C recombinant polypeptide that allows direct delivery of the peptide into the cells showed reduced expression of HCV IRES activity in this cell line. The study reveals valuable insights into the role of La protein in ribosome assembly at the HCV IRES and also provides the basis for targeting ribosome-HCV IRES interaction to design potent antiviral therapy.

Amino Acid Motifs↗
PubMed2005

Factor-independent assembly of elongation-competent ribosomes by an internal ribosome entry site located in an RNA virus that infects penaeid shrimp.

The Taura syndrome virus (TSV), a member of the Dicistroviridae family of viruses, is a single-stranded positive-sense RNA virus which contains two nonoverlapping reading frames separated by a 230-nucleotide intergenic region. This intergenic region contains an internal ribosome entry site (IRES) which directs the synthesis of the TSV capsid proteins. Unlike other dicistroviruses, the TSV IRES contains an AUG codon that is in frame with the capsid region, suggesting that the IRES initiates translation at this AUG codon by using initiator tRNAmet. We show here that the TSV IRES does not use this or any other AUG codon to initiate translation. Like the IRES in cricket paralysis virus (CrPV), the TSV IRES can assemble 80S ribosomes in the absence of initiation factors and can direct protein synthesis in a reconstituted system that contains only purified ribosomal subunits, eukaryotic elongation factors 1A and 2, and aminoacylated tRNAs. The functional conservation of the CrPV-like IRES elements in viruses that can infect different invertebrate hosts suggests that initiation at non-AUG codons by an initiation factor-independent mechanism may be more prevalent.

Animals↗
PubMed1991

Ribosomal protein genes are overexpressed in colorectal cancer: isolation of a cDNA clone encoding the human S3 ribosomal protein.

We have isolated a cDNA clone encoding the human S3 ribosomal protein from a normal human colon cDNA library. The clone was identified as one of many that detected genes whose level of expression was increased in adenocarcinoma of the colon relative to normal colonic mucosa. Increased levels of the S3 transcript were present in the tumors of all eight patients examined. Moreover, the S3 mRNA was also more abundant in 7 of 10 adenomatous polyps, the presumed precursor of carcinoma. Additional studies demonstrated that increased levels of mRNAs encoding several other ribosomal proteins, including S6, S8, S12, L5, and P0, were present in colorectal tumors and polyps. These results suggest that there is increased synthesis of ribosomes in colorectal tumors and that this increase is an early event in colon neoplasia.

Adenocarcinoma↗
PubMed1991

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

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

Amino Acid Sequence↗
PubMed2006

The other target for ribosomal antibiotics: inhibition of bacterial ribosomal subunit formation.

The development of microbial resistance to practically all currently used antimicrobial agents has spurred efforts to develop new antibiotics and to identify novel targets in bacterial cells. This review summarizes the evidence for inhibition of bacterial ribosomal subunit formation as a target for many antibiotics distinct from their well-known inhibition of translation. Features of a model to explain this activity are explored. Results are presented to show the accumulation of both 30S and 50S ribosomal subunit precursors in antibiotic inhibited cells. These precursors have been characterized and are shown to bind radio-labeled drugs. Pulse and chase labeling studies have revealed the slower rates of subunit synthesis in drug treated cells compared with uninhibited controls. Resynthesis of subunits after antibiotic removal precedes recovery of control protein synthesis capacity, consistent with the model presented. Also certain mutant strains defective in different ribonuclease activities are more susceptible to antibiotic inhibition of assembly as predicted. Results indicating the equivalence of assembly inhibition and translational inhibition are described. Lastly, the identification of a 50S subunit precursor particle as a substrate for rRNA methyltransferase activity is shown. The weight of evidence presented clearly indicates that ribosomal antibiotics have a second target in cells. Inhibition of cell growth and subsequent cell death results from the activity of these antibiotics on the combined targets. The possibility of designing assembly specific inhibitors is discussed.

Aminoglycosides↗