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At least 289 records · Page 16Linked to original sources
PubMed1994

Correlation between the activities of five ribosome-inactivating proteins in depurination of tobacco ribosomes and inhibition of tobacco mosaic virus infection.

The rRNA depurination activities of five ribosome-inactivating proteins (RIPs) were compared in vitro using yeast and tobacco leaf ribosomes as substrates. All of the RIPs (pokeweed antiviral protein (PAP), dianthin 32, tritin, barley RIP and ricin A-chain) were active on yeast ribosomes. PAP and dianthin 32 were highly active and ricin A-chain weakly active on tobacco ribosomes, whereas tritin and barley RIP were inactive. PAP and dianthin 32 were highly effective in inhibiting the formation of local lesions caused by tobacco mosaic virus (TMV) on tobacco leaves, whereas tritin, barley RIP and ricin A-chain were ineffective. The apparent anomaly between the in vitro rRNA depurination activity, but lack of antiviral activity of ricin A-chain was further investigated by assaying for rRNA depurination in situ following the topical application of the RIP to leaves. No activity was detected, a finding consistent with the apparent lack of antiviral activity of this RIP. Thus, it is concluded that there is a positive correlation between RIP-catalysed depurination of tobacco ribosomes and antiviral activity which gives strong support to the hypothesis that the antiviral activity of RIPs works through ribosome inactivation.

Aniline Compounds↗
PubMed1988

Reversion from erythromycin dependence in Escherichia coli: strains altered in ribosomal sub-unit association and ribosome assembly.

A mutant of Escherichia coli dependent on erythromycin for growth spontaneously gives erythromycin-independent strains with altered or missing ribosomal proteins. strains with defects in ribosome assembly were sought and obtained from among these revertants. Two organisms in which ribosomal protein L19 is altered and absent respectively have 70S ribosomes whose dissociation into sub-units is particularly sensitive to pressures generated during centrifuging. The mutant that lacks protein L19 also accumulates ribosome precursor particles during exponential growth as do others including mutants that lack proteins S20 or L1. These strains also show unbalanced synthesis of RNA and so will be useful in investigating both the pathways and the regulation of ribosome assembly.

Bacterial Proteins↗
PubMed1982

The properties of the tRNA . protein complex of the Escherichia coli ribosome. Interaction with tRNA, 5-S RNA and 30-S ribosomal subunit.

A complex between immobilized tRNA and Escherichia coli 50-S ribosomal subunit proteins provides binding sites for several other components of the protein biosynthesis system. We found that this complex binds 5-S RNA. tRNA and 30-S ribosomal subunits. The binding of 5-S RNA and tRNA takes place at equilibrium; in 10 mM Tris/HCl buffer pH 7.5, containing 10 mM MgCl2 and 100 mM KCl at 4 degrees C it is characterized by the dissociation constants 1.7 X 10(-7) M and 4 X 10(-7) M respectively. We show that the performed complex between tRNA and 50-S ribosomal subunit protein can bind these two RNAs simultaneously by forming a subribosomal domain consisting of two molecules of tRNA, 5-S RNA and the ribosomal proteins L2, L15, L16, L17, L18, L22, L33 and L34. The preformed tRNA . protein complex interacts also with the 30-S ribosomal subunit, suggesting that is located at the ribosomal interface.

Escherichia coli↗
PubMed2005

Accessibility of 18S rRNA in human 40S subunits and 80S ribosomes at physiological magnesium ion concentrations--implications for the study of ribosome dynamics.

Protein biosynthesis requires numerous conformational rearrangements within the ribosome. The structural core of the ribosome is composed of RNA and is therefore dependent on counterions such as magnesium ions for function. Many steps of translation can be compromised or inhibited if the concentration of Mg(2+) is too low or too high. Conditions previously used to probe the conformation of the mammalian ribosome in vitro used high Mg(2+) concentrations that we find completely inhibit translation in vitro. We have therefore probed the conformation of the small ribosomal subunit in low concentrations of Mg(2+) that support translation in vitro and compared it with the conformation of the 40S subunit at high Mg(2+) concentrations. In low Mg(2+) concentrations, we find significantly more changes in chemical probe accessibility in the 40S subunit due to subunit association or binding of the hepatitis C internal ribosomal entry site (HCV IRES) than had been observed before. These results suggest that the ribosome is more dynamic in its functional state than previously appreciated.

Humans↗
PubMed1982

Identification by RNA-protein cross-linking of ribosomal proteins located at the interface between the small and the large subunits of mammalian ribosomes.

Protein constituents at the subunit interface of rat liver ribosomes were analysed by cross-linking with the bifunctional reagent, diepoxybutane (distance between reactive groups 4 A). Isolated 40S and 60S subunits were labelled with 125I and recombined with unlabelled complementary subunits. The two kinds of selectively labelled 80S ribosomes were treated with diepoxybutane at low concentration. Radioactive ribosomal proteins covalently attached to the rRNA of the unlabelled complementary subparticles were isolated by repeated gradient centrifugation. The RNA-bound, labelled proteins were identified by two-dimensional gel electrophoresis. The experiments showed that proteins S2, S3, S4, S6, S7, S13, and S14 in the small subunit of rat liver ribosomes are located at the ribosomal interface in close proximity to 28S rRNA. Similarly, proteins L3, L6, L7, and L8 were found at the the interface of the large ribosomal subunit in the close vicinity of 18S rRNA.

Animals↗
PubMed2001

Ray38p, a homolog of a purine motif triple-helical DNA-binding protein, Stm1p, is a ribosome-associated protein and dissociated from ribosomes prior to the induction of cycloheximide resistance in Candida maltosa.

Cycloheximide (CYH) resistance in Candida maltosa is dependent on the induction of a ribosomal protein, Q-type L41, the 56th residue of which is glutamine, not proline as in ordinary P-type L41. We found that a 38-kDa protein in a wild-type C. maltosa ribosomal fraction became undetectable upon CYH treatment but detectable again with the establishment of CYH resistance by the induction of Q-type L41. We cloned a gene coding for this protein and named it RAY38 (ribosome-associated protein of yeast). Ray38p is a homolog of a purine motif triple-helical DNA-binding protein, Stm1p, and has a putative RNA-binding motif RGG. The ribosome-associated Ray38p was phosphorylated at serine and threonine residues, and Ray38p that was dissociated from ribosome by CYH treatment was highly phosphorylated in threonine residues. A ray38 null mutant recovered faster from CYH-caused growth stasis than the wild-type strain, suggesting that the dissociation of Ray38p from ribosome facilitates the induction of CYH resistance in C. maltosa.

Amino Acid Motifs↗
PubMed1993

Two inactive fragments derived from the yeast mitochondrial ribosomal protein MrpS28 function in trans to support ribosome assembly and respiratory growth.

The mitochondrial ribosomal protein MrpS28 of Saccharomyces cerevisiae is one of several mitochondrial ribosomal proteins homologous to Escherichia coli ribosomal proteins within the context of a larger protein. Relative to a region of homology with E. coli ribosomal protein S15, the mature MrpS28 protein has unique sequence domains of 117 and 48 amino acids at its amino and carboxyl terminus, respectively. To better understand the role of the various sequence domains of the MrpS28 protein in vivo, truncated derivatives were expressed under conditions where they were the only potential source of functional MrpS28 protein. The results shown here demonstrate that the amino-terminal domain and the S15-like domain are both essential for respiratory growth. Interestingly an inactive amino-terminal fragment can be complemented in trans by a second inactive fragment comprising the S15-like domain and the carboxyl-terminal 48 amino acids. Consequently, the assembly of these fragments into ribosomal subunits can be examined when they are expressed individually or together. Results from these studies indicate that each of the MrpS28-derived fragments facilitates the incorporation of the other into 37 S ribosomal subunits.

Amino Acid Sequence↗
PubMed1977

Interaction of the cytoplasmic membrane and ribosomes in Escherichia coli; altered ribosomal proteins in sucrose-dependent spectinomycin-resistant mutants.

Alterations in the ribosomes of sucrose-dependent spectinomycin-resistant (Sucd-Spcr) mutants of Escherichia coli were studied. Subunit exchange experiments showed that 30S subunits were responsible for the resistance of ribosomes to spectinomycin in all Sucd-Spcr mutants tested. Proteins of 30S ribosomes were analyzed by carboxymethyl cellulose column chromatography based on their elution positions. Mutants YM22 and YM93 had an altered 30S ribosomal protein component, S5, and mutant YM50 had an altered protein, S4. Although a shift of elution position was not detected for all the 30S ribosomal proteins from mutant YM101, the amount of protein S3 was appreciably lowered in the isolated 30S subunits. A partial reconstitution experiment with protein S3 prepared from both the wild-type strain and YM101 revealed that the mutant had altered protein S3 which is responsible for the spectinomycin resistance. These alterations in 30S subunits are discussed in relation to the interaction between ribosomes and the cytoplasmic membrane.

Cell Membrane↗
PubMed1993

A nuclear mutation conferring thiostrepton resistance in Chlamydomonas reinhardtii affects a chloroplast ribosomal protein related to Escherichia coli ribosomal protein L11.

We have isolated a nuclear mutant (tsp-1) of Chlamydomonas reinhardtii which is resistant to thiostrepton, an antibiotic that blocks bacterial protein synthesis. The tsp-1 mutant grows slowly in the presence or absence of thiostrepton, and its chloroplast ribosomes, although resistant to the drug, are less active than chloroplast ribosomes from the wild type. Chloroplast ribosomal protein L-23 was not detected on stained gels or immunoblots of total large subunit proteins from tsp-1 probed with antibody to the wild-type L-23 protein from C. reinhardtii. Immunoprecipitation of proteins from pulse-labeled cells showed that tsp-1 synthesizes small amounts of L-23 and that the mutant protein is stable during a 90 min chase. Therefore the tsp-1 phenotype is best explained by assuming that the mutant protein synthesized is unable to assemble into the large subunit of the chloroplast ribosome and hence is degraded over time. L-23 antibodies cross-react with Escherichia coli r-protein L11, which is known to be a component of the GTPase center of the 50S ribosomal subunit. Thiostrepton-resistant mutants of Bacillus megaterium and B. subtilis lack L11, show reduced ribosome activity, and have slow growth rates. Similarities between the thiostrepton-resistant mutants of bacteria and C. reinhardtii and the immunological relatedness of Chlamydomonas L-23 to E. coli L11 suggest that L-23 is functionally homologous to the bacterial r-protein L11.

Animals↗
PubMed1975

Localization of Escherichia coli ribosomal protein S4 on the surface of the 30S ribosomal subunit by immuno electron microscopy. I. Distribution of antibody-binding sites as obtained with immunoglobulins and monovalent antibody fragments from various S4-specific antisera.

The location of the ribosomal protein S4 on the surface of the 30S subunit of E. coli ribosomes was determined by immuno electron microscopy. Immunoglobulins from six separate S4-specific antisera were investigated. In accordance with earlier findings protein S4 was shown to have an elongated conformation within the antisera. Protein S4 has therefore definitely an extended or fibrous shape in the intact ribosome (Lake et al., 1974; Stöffler and Tischendorf, 1975; Tischendorf et al., 1975). S4 specific antibodies bind at four distinct sites of 30S ribosomal subunits, designated A, B, C and D. Two antibody binding sites (A and B) are located on the "head", they were shown to be separated by 70-85 A. The distance between these sites and the two sites C and D on the "body" of the subunit amounts to at least 90-125 A; hence protein S4 should be extended to a total length of approximately 160-200 A. At least three of the four S4-specific antibody binding sites were observed with antibodies from each of the six investigated. These sites were observed independently of whether isolated S4 protein, an S4-16S rRNA complex or 30S ribosomes were used as the antigen. They could also be visualized with monovalent antibody fragments (Fab). S4-specific antibodies enriched by affinity chromatography bound at identical sites which conclusively ensures that antibody binding at these sites is specific for protein S4 and is not due to the presence of contaminating antibodies in the intact ribosome. The implications of these results with respect of the correlation of each of the sites to their respective antigenically active fragments within protein S4 are discussed.

Antigens, Bacterial↗
PubMed1977

Isolation and characterization of cytoplasmic and chloroplastic ribosomes and their ribosomal RNAs from the diatom Cylindrotheca fusiformis.

The cytoplasmic and chloroplast ribosomes from the marine diatom Cylindrotheca fusiformis were isolated and characterized. The cytoplasmic ribosomes sedimented in sucrose at 84S and dissociated into subunits of 64S and 42S in the absence of Mg2+. It contained ribosomal RNAs with molecular weights of 1.31 X 10(6) and 0.70 X 10(6). The chloroplast ribosomes sedimented at 70S only in the presence of high Mg2+ concentrations (25-100 mM). No stable subunits were routinely observed and at very high levels of Mg2+ (greater than 100 mM) the 70S species was converted to a form sedimenting at 55S. At 4 degrees C ribosomal RNAs with molecular weights of 1.1 X 10(6) and 0.40 X 10(6) were detected on polyacrylamide gel electrophoresis. When the RNAs were resolved at room temperature the large molecular weight component disappeared while RNA with molecular weights of 0.65 X 10(6) and 0.53 X 10(6) were observed. Apparently the large chloroplast RNAs dissociated into two pieces of unequal molecular weight. These properties of the diatom's chloroplast ribosomes are very similar to those of the counterparts in unicellular green algae, which suggests that both types of algae have a common phylogenetic ancestor.

Biological Evolution↗
PubMed2005

Human ribosomal protein L18a interacts with hepatitis C virus internal ribosome entry site.

Translation initiation of hepatitis C virus RNA occurs via ribosome binding to an 'internal ribosome entry site (IRES)' located in the 5'untranslated region of the viral RNA. The principle interaction between the 40S ribosomal subunit and the HCV IRES has been shown to be largely factor independent, which is followed by the joining of the 60S ribosomal subunit to form functional 80S complex. However several additional cellular proteins have been reported to bind to HCV IRES and enhance the initiation of translation. In order to identify novel factors involved in the ribosome assembly during internal initiation of HCV RNA, northwestern screening of a HeLa cDNA expression library was performed, using HCV IRES RNA as probe. We demonstrate here, that human ribosomal protein L18a, a constituent of 60S subunit, interacts with HCV IRES RNA. This interaction was further confirmed by using a recombinant protein similar to L18a (sL18a), cloned from human blood. Interestingly, addition of increasing concentration of the purified recombinant sL18a protein, showed moderate stimulation of HCV IRES activity in the in vitro translation assay. These observations suggest that the human L18a might influence the HCV IRES mediated translation.

5' Untranslated Regions↗
PubMed1981

The protein synthetic activity in vitro of ribosomes differing in the extent of phosphorylation of their ribosomal proteins.

We describe a re-examination of the cell-free protein synthetic activity of eukaryotic ribosomes having proteins phosphorylated to different extents. Ribosomal 40 S subunits were isolated both from a variety of cells in which there is relatively little phosphorylation of ribosomal protein S6, and from cells subjected in vivo to different stimuli that promote the extensive phosphorylation of protein S6. The ability of these subunits to bind Met-tRNA as well as the second amino acyl-tRNA (Val-tRNA) was compared in the presence of highly purified initiation factors, elongation factor EF-1 at various concentrations of 60S subunits, 9 S globin mRNA and potassium ions. The ability of the subunits to synthesize polyphenylalanine was also studied using highly purified elongation factors. In no case was any significant difference in activity observed between ribosomes with protein S6 phosphorylated to different extents. Similar, though less extensive, studies were preformed comparing 60 S ribosomal subunits differing in the extent of phosphorylation of the acidic phosphoprotein, L gamma , and of L14. No difference in activity was observed between these ribosomes.

Animals↗
PubMed1992

Differential requirement of ATP and extra-ribosomal proteins for ribosome inactivation by eight RNA N-glycosidases.

The requirement of ATP and extra-ribosomal proteins for the inactivation of ribosomes by eight plant RNA N-glycosidases [ribosome-inactivating proteins (RIPs)] was investigated. Tritin, pokeweed antiviral protein and barley RIP depend, as gelonin [Sperti, S., Brigotti, M., Zamboni, M., Carnicelli, D. and Montanaro, L. (1991) Biochem. J., 277, 281-284], on the presence of ATP and extra-ribosomal proteins for full inactivation of ribosomes, while bryodin, lychnin, momordin, momorcochin and saporin inactivate isolated Artemia salina ribosomes suspended in buffer saline.

Adenosine Triphosphate↗
PubMed1991

Localization of surface peptide from ribosomal protein L7 on 80 S ribosome by biotinylation.

A surface topography of ribosomal peptides on ribosome particles was conducted by using N',Hydroxysuccinimido-biotin (NHS-biotin) modification. All rat ribosomal proteins, except proteins L3 and L8, are biotinylated when the ribosome particle is the substrate. A surface peptide from protein L7 was determined from biotinylated ribosomes by high performance liquid chromatography and cyanogen bromide peptide mapping. It was found that only the tandem repeats of the NH2-terminal segment of protein L7 are accessible to biotinylation. It is concluded that the NH2-terminal-end of protein L7 should be exposed on the surface of ribosomal particles.

Animals↗
PubMed1982

Effects of ethionine treatment of protein-synthesizing apparatus of rat liver 80 S ribosomes and 40 S ribosomal subunits.

The inhibitory effects of ethionine treatment of female rats for 4 h on the protein-synthesizing machineries of 80 S ribosomes and 40 S ribosomal subunits of the liver were investigated. The following results were obtained. (1) The translation of globin mRNA by 80 S ribosomes or 40 S ribosomal subunits, in combination with mouse 60 S subunits, was markedly inhibited by ethionine treatment in a complete cell-free system containing partially purified initiation factors of rabbit reticulocytes and the rat liver pH 5 fraction. (2) The polysome formation of 80 S ribosomes in the complete system described above was inhibited by ethionine treatment. Similar inhibitions by ethionine treatment were observed in the case of incubation of 40 S subunits with reticulocyte lysate, although the polysome formation was rather low even in the case of control 40 S subunits. (3) The pattern of CsCl isopycnic centrifugation of rat liver native 40 S subunits uniformly labeled with [14C]- or [3H]orotic acid showed that the content of non-ribosomal proteins of native 40 S subunits was decreased by ethionine treatment. The analysis of proteins of native 40 subunits by SDS-polyacrylamide slab gel electrophoresis revealed that eIF-3 subunits and two unidentified protein fractions of molecular weight of 2.3.10(4) and 2.1.10(4) were decreased in ethionine-treated rate liver. (4) 40 S subunits from ethionine-treated or control rat livers were labeled with N-[3H]ethylmaleimide or N-[14C]ethylmaleimide, and the 3H to 14C ratios of individual 40 S proteins on two-dimensional polyacrylamide gel electrophoresis were measured. The results suggested that the conformation of rat liver 40 S subunits was changed by ethionine treatment. (5) These results may indicate that ethionine treatment decreases the activity of rat liver 40 S subunits for the interaction with initiation factors, especially eIF-3, as the results of conformational changes of 40 S subunits.

Animals↗
PubMed1990

Site-specific mutation of the conserved m6(2)A m6(2)A residues of E. coli 16S ribosomal RNA. Effects on ribosome function and activity of the ksgA methyltransferase.

In vitro synthesis of mutant 16S RNA and reconstitution with ribosomal proteins into a mutant 30S ribosome was used to make all possible single base changes at the universally conserved A1518 and A1519 residues. All of the mutant RNAs could be assembled into a ribosomal subunit which sedimented at 30 S and did not lack any of the ribosomal proteins. A series of in vitro tests of protein synthesis ability showed that all of the mutants had some activity. The amount varied according to the assay and mutant, but was never less than 30% and was generally above 50%. Therefore, neither the conserved A1518 nor A1519 residues are essential for ribosome function. The mutant ribosomes could also be methylated by the ksgA methyltransferase to 70-120% of the expected amount. Thus, neither of the A residues is required for methylation of the other, ruling out any obligate order of methylation of A1518 and A1519.

Adenine Nucleotides↗
PubMed1990

Nuclease S1 mapping of 16S ribosomal RNA in ribosomes.

Escherichia coli 16S rRNA and 16S-like rRNAs from other species have several universally conserved sequences which are believed to be single-stranded in ribosomes. The quantitative disposition of these sequences within ribosomes is not known. Here we describe experiments designed to explore the availability of universal 16S rRNA sequences for hybridization with DNA probes in 30S particles and 70S ribosomes. Unlike previous investigations, quantitative data on the accessibility of DNA probes to the conserved portions of 16S rRNA within ribosomes was acquired. Uniquely, the experimental design also permitted investigation of cooperative interactions involving portions of conserved 16S rRNA. The basic strategy employed ribosomes, 30S subunits, and 16S rRNAs, which were quantitatively analyzed for hybridization efficiency with synthetic DNA in combination with nuclease S1. In deproteinated E. coli 16S rRNA and 30S subunits, the regions 520-530, 1396-1404, 1493-1504, and 1533-1542 are all single-stranded and unrestricted for hybridization to short synthetic DNAs. However, the quantitative disposition of the sequences in 70S ribosomes varies with each position. In 30S subunits there appear to be no cooperative interactions between the 16S rRNA universal sequences investigated.

Base Sequence↗