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1.
《Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis》1976,418(3):397-403
The phosphorylation of ribosomal proteins from eukaryotes in homologous and heterologous cell-free systems has been studied. The ribosomes and protein kinases from yeast (Saccharomyces cerevisiae, strain Bu), wheat (Triticum vulgare) and rabbit (Orystolagus cuniculus) have been used.It has been found that five ribosomal proteins incorporate γ-32P from ATP during the incubation of wheat ribosomes with wheat protein kinase. When the phosphorylation of isolated wheat ribosomal proteins was examined more phosphoproteins were detected. These data confirm the suggestion that the ribosomal structure affects the phosphorylation. Probably some ribosomal proteins remain hidden for the action of protein kinase.The results from the crossed experiments show that there is no barrier for phosphorylation of yeast ribosomes with liver protein kinase, of wheat ribosomes with yeast and liver protein kinases and of liver ribosomes with yeast and plant protein kinases. The wheat protein kinase does not phosphorylate the yeast ribosomes under these experimental conditions. Some differences in the set of phosphoproteins obtained with various protein kinases have been detected. These data suggest that the ribosomal protein phosphorylation is not highly species specific although it is not universal. 相似文献
2.
The Mg2+ precipitation method has been adapted for isolation of ribosomes from roots of wheat. The ribosomes prepared by this procedure show A260/A280 = 1.6 and A260/A235 = 1.3 and contain 44d% RNA and 56% ribosomal proteins. There are no detectable differences in the ribosomal protein complement and accessibility of the ribosomal proteins to phosphorylation between ribosomes isolated by this procedure and those prepared by classical ultracentrifugation methods. The ribosomes are active in a poly-U directed cell-free system for protein synthesis. 相似文献
3.
Adaptive Potential of Wheat Ribosomes toward Heat Depends on the Large Ribosomal Subunit and Ribosomal Protein Phosphorylation 总被引:1,自引:1,他引:0
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In a study of the translational efficiency of ribosomal subunits as a function of an in vivo temperature pretreatment, ribosomes were isolated from heat-pretreated (36°C) and reference (20°C) wheat seedlings (Triticum aestivum L.). The efficiency of recombined subunits in translating polyuridylic acid was assessed. A threefold increase in the rate of incorporation of phenylalanine by ribosomes from heat-pretreated plants was due to the large ribosomal subunit. This adaptive temperature effect was not correlated with a higher thermal stability of ribosomes or subunits from heat-pretreated seedlings, and two-dimensional gel electrophoresis failed to detect structural alterations of ribosomal proteins. Phosphorylation of ribosomal proteins in vitro showed no differences between ribosomes or subunits from heat-pretreated and reference plants. Incubation with [32P]orthophosphate in vivo led to twice the amount of phosphate in ribosomal proteins from heat-pretreated wheat seedlings. This result is important with respect to the evaluation of the molecular basis of enhanced translational efficiency of ribosomes isolated from heat-pretreated wheat seedlings. 相似文献
4.
Membrane-bound ribosomes were isolated from a post-mitochondrial supernatant fraction of mouse liver homogenate by sedimentation in a sucrose density gradient. Loose ribosomes were released from the membrane fragments with 0.5 M KCl, while tight bound ribosomes were not released. After purification of the loose and tight ribosomes subclasses, ribosomal subunit proteins were isolated and compared by two-dimensional polyacrylamide gel electrophoresis. No differences in the ribosomal protein composition was detected. 相似文献
5.
The Chloroplast and Cytoplasmic Ribosomes of Euglena: II. Characterization of Ribosomal Proteins
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Cytoplasmic and chloroplast ribosomal proteins were isolated from Euglena gracilis and analyzed on polyacrylamide gels. Cytoplasmic ribosomes appear to contain 75 to 100 proteins ranging in molecular weight from 10,200 to 104,000, while chloroplast ribosomes appear to contain 35 to 42 proteins with molecular weights ranging from 9,700 to 57,900. This indicates that the cytoplasmic ribosomes are similar in composition to other eucaryotic ribosomes, while chloroplast ribosomes have a protein composition similar to the 70S procaryotic ribosome. The kinetics of light-induced labeling of cytoplasmic ribosomal proteins during chloroplast development has been determined, and the results are compared with the kinetics of ribosomal RNA synthesis. 相似文献
6.
Cytoplasmic monoribosomes from freshly cut and ‘aged’ carrot root disks were characterized relative to the Mg2+ optima for poly U (polyuridylic acid)-directed phenylalanine incorporation, the ease of dissociation by KCl in the presence of Mg2+, the ability to bind 3H-poly U, and acrylamide gel fractionation of the ribosomal proteins. The differences in in vitro amino acid incorporation by ribosomes and supernatant from fresh and ‘aged’ disks were confined to the ribosome fraction. The Mg2+ optima for poly U-directed 14C-phenylalanine incorporation was 16 mM for ribosomes from ‘aged’ disks compared to 20 mM for ribosomes from fresh disks. Monoribosomes from the fresh disks were easily dissociated into subunits (0·2 M KCl in 5 mM Mg2+) while the ribosomes from ‘aged’ disks were not completely dissociated even in 0·5 M KCl. Ribosomes from ‘aged’ disks were more effective in binding 3H-poly U than ribosomes from fresh disks. When the disks were subjected to an anaerobic environment prior to ribosome extraction (to strip monoribosomes of peptidyl-t RNA) the above effects of ‘aging’ were reversed. These results suggest that increased monoribosome activity associated with ‘aging’ may be related in part to an increase in the level of peptidyl-tRNA associated with the ribosomes. Acrylamide gel electrophoresis profiles of ribosomal proteins extracted from ribosomes of fresh and ‘aged’ tissue suggest that a change in the protein complement may also be important to the observed changes in ribosomal activity. The ribosomes from ‘aged’ disks contained at least two components not associated with ribosomes from fresh disks. 相似文献
7.
María Rodríguez-Mateos David Abia Juan J. García-Gmez Antonio Morreale Jesús de la Cruz Cruz Santos Miguel Remacha Juan P. G. Ballesta 《Nucleic acids research》2009,37(11):3514-3521
In Saccharomyces cerevisiae, the Mrt4 protein is a component of the ribosome assembly machinery that shares notable sequence homology to the P0 ribosomal stalk protein. Here, we show that these proteins can not bind simultaneously to ribosomes and moreover, a chimera containing the first 137 amino acids of Mrt4 and the last 190 amino acids from P0 can partially complement the absence of the ribosomal protein in a conditional P0 null mutant. This chimera is associated with ribosomes isolated from this strain when grown under restrictive conditions, although its binding is weaker than that of P0. These ribosomes contain less P1 and P2 proteins, the other ribosomal stalk components. Similarly, the interaction of the L12 protein, a stalk base component, is affected by the presence of the chimera. These results indicate that Mrt4 and P0 bind to the same site in the 25S rRNA. Indeed, molecular dynamics simulations using modelled Mrt4 and P0 complexes provide further evidence that both proteins bind similarly to rRNA, although their interaction with L12 displays notable differences. Together, these data support the participation of the Mrt4 protein in the assembly of the P0 protein into the ribosome and probably, that also of the L12 protein. 相似文献
8.
John E. Kinsella 《Biochimica et Biophysica Acta (BBA)/General Subjects》1984,801(3):416-423
Exposure of yeast 80 S ribosomes to chaotropic salts such as NaClO4 or NaSCN at concentrations as low as 0.4 M resulted in complete dissociation and subsequent aggregation of the ribosomal proteins. However, under similar conditions, both NaCl and NaBr did not cause dissociation and aggregation. The protein precipitate obtained by exposing the ribosomes to 0.5 M NaClO4 was free of any rRNA contamination as judged by ultraviolet-absorption analysis. Comparison of the two-dimensional polyacrylamide gel electrophoretic analysis of the above ribosomal protein precipitate with that ribosomal proteins isolated by the standard acetic acid extraction procedure revealed that the protein precipitate contained all the ribosomal proteins. Based on these results, a simple method for the isolation of total ribosomal proteins and rRNA under mild, nondenaturing conditions is proposed. A possible mechanism for the dissociation of proteins from the ribosome by chaotropic salts is also discussed. 相似文献
9.
《Insect Biochemistry》1985,15(3):391-401
Stimulation of the accessory gland of Drosophila melanogaster males by copulation was studied at the molecular level by in vivo radiolabelling experiments. In addition, experiments were carried out to clarify the molecular basis of regulation of the gland's synthetic activityThe stimulatory effect of copulation on secretion protein synthesis is observed within minutes after copulation is terminated. Repeated copulations elicit a stronger response. The data indicate a coordinate synthesis of the secretory proteins.In a reticulocyte-lysate system with added total RNA extract from male accessory glands, secretion proteins appear as translation products. It is shown that synthesis of secretory protein messages is not enhanced by copulation and that the messages are stable.Synthesis of ribosomal proteins and ribosomal RNA increases upon copulation. The increase is observed in the time period from 1 to 5 hr after copulation. Again, the synthesis of the various ribosomal proteins is well coordinated. There is no elevation in the accumulation of ribosomal protein mRNAs after copulation. 相似文献
10.
Andrew G. Lambertsson S. Bertil Rasmuson Gunnar D. Bloom 《Molecular & general genetics : MGG》1970,108(4):349-357
Summary Ribosomes were isolated from larvae and adult flies, and the purity of the preparation was checked by electron microscopy. The ribosomal proteins were extracted with cold dilute hydrochloric acid, and precipitated with cold acetone. The proteins were characterized by polyacrylamide gel electrophoresis. At pH 3.0 at least 25 bands of different color intensities were resolved, forming a complex pattern.On the basis of electrophoretic mobilities, it was shown that some ribosomal proteins are species-specific, and that larval ribosomes have three protein components more than ribosomes from adult flies.Incubation of the ribosomes with 0.75 M NH4Cl at a low Mg++ concentration lead to detachment of 64% of the ribosomal protein. This detachment of protein molecules was considerably reduced by a five-fold increase of Mg++ ions. 相似文献
11.
The proteins associated with the ribosomal subunits having the molecular masses from 158 to 47 kDa were isolated from hyaloplasmic, nucleoid and membrane fractions of Escherichia coli MRE600 cells. The proteins are eliminated from 50S subunits of ribosomes by thrice washing with the 1 M ammonium chloride buffer. 50S subunit proteins were found to be immunologically related to the inner membrane proteins. The native 50S subunits of ribosomes possess the expressed ATP-ase activity, while the washed off subunits lose it completely. 相似文献
12.
Rat liver ribosomes were dissociated into subunits using EDTA, sodium pyrophosphate, high concentrations of KC1, as well as by incubation with puromycin in presence of 0.5 M KC1. The subunits obtained were analyzed using the density gradient centrifugation technique and their ribosomal proteins were separated by means of two-dimensional polyacrylamide gel electrophoresis. The ribosomal protein patterns of the two subunits isolated using each of the dissociating method were compared to the protein patterns of monosomes prepared by puromycin treatment alone. Our results revealed that the use of chelating agents to dissociate the ribosomes resulted in the loss of some ribosomal proteins from the small subunit. On the other hand, the use of KC1 in high concentrations to dissociate the ribosomes did not appear to cause any major loss of proteins from the ribosomes except for some acidic proteins. 相似文献
13.
The amino acid composition of overall protein of ribosomes and ribosomal subunits of pea seeds has been found typical of ribosomal protein. Electrophoresis in polyacrylamide gel demonstrates that proteins extracted by the solution of 3 M LiCl-4 M urea from purified ribosomes of pea seeds move towards the cathode at pH 2.2 and separate into 41 components. Electrophoresis in a tris-glycine buffer at pH 9.2 does not reveal any substance corresponding to acid proteins. Similar distribution patterns are observed when ribosomal particles are isolated with or without triton (0,5%). The treatment of ribosomes by deoxycholate results in some changes, depending on the detergent concentration. All the protein components detected in ribosomes, except one, are present in the subunits. Proteins of large and small ribosome subunits produced 26 and 21 components respectively in polyacrylamide gel electrophoresis. The distribution patterns of proteins of the two subunits appear to be different. The majority of the components of the large and small subunits differ in mobility. The data obtained suggest considerable specificity of the protein composition of 60S and 40S subunits of 80S ribosomes in higher plants. 相似文献
14.
The induction of antituberculous immunity by crude ribosomal fractions isolated from Mycobacterium tuberculosis strain H37Ra, M. bovis strain BCG, and M. smegmatis was studied in CF-1 mice. Levels of antituberculous immunity similar to that induced by live BCG were induced by the BCG and H37Ra ribosomal fractions whereas that isolated from M. smegmatis was found to be inactive. Electrophoresis of the three ribosomal fractions in sodium dodecyl sulfate - polyacylamide gels followed by differential staining showed the two active ribosomal fractions to be similar in their proteins, carbohydrate-containing substances, and lipid profiles. The inactive smegmatis ribosomal fraction differed mainly from the active ones on the basis of its carbohydrate-containing substances profile and by the absence of lipids. The polysaccharides and the ribosomes present in the H37Ra ribosomal fractions were purified by affinity chromatography on concanavalin A - Sepharose 4B. Each purified preparation showed no or only low antituberculous activity when injected separately, but when mixed together a high protection was observed. The formation of complexes between the ribosomes and the polysaccharide fraction was suggested and appears to be necessary for the induction of antituberculous immunity. 相似文献
15.
The amount of chloroplast ribosomal RNAs of Chlamydomonas reinhardtii which sediment at 15,000 g is increased when cells are treated with chloramphenicol. Preparations of chloroplast membranes from chloramphenicol-treated cells contain more chloroplast ribosomal RNAs than preparations from untreated cells. The membranes from treated cells also contain more ribosome-like particles, some of which appear in polysome-like arrangements. About 50% of chloroplast ribosomes are released from membranes in vitro as subunits by 1 mM puromycin in 500 mM KCl. A portion of chloroplast ribosomal subunits is released by 500 mM KCl alone, a portion by 1 mM puromycin alone, and a portion by 1 mM puromycin in 500 mM KCl. Ribosomes are not released from isolated membranes by treatment with ribonuclease. Membranes in chloroplasts of chloramphenicol-treated cells show many ribosomes associated with membranes, some of which are present in polysome-like arrangements. This type of organization is less frequent in chloroplasts of untreated cells. Streptogramin, an inhibitor of initiation, prevents chloramphenicol from acting to permit isolation of membrane-bound ribosomes. Membrane-bound chloroplast ribosomes are probably a normal component of actively growing cells. The ability to isolate membrane-bound ribosomes from chloramphenicol-treated cells is probably due to chloramphenicol-prevented completion of nascent chains during harvesting of cells. Since chloroplasts synthesize some of their membrane proteins, and a portion of chloroplast ribosomes is bound to chloroplast membranes through nascent protein chains, it is suggested that the membrane-bound ribosomes are synthesizing membrane protein. 相似文献
16.
Summary The ribosomal subunit proteins (30S and 50S) from vegetative and sporulating cells of Bacillus subtilis 168M were analyzed by two dimensional acrylamide gel electrophoresis. Twenty two proteins were identified in the 30S subunits and 28 proteins are detectable in the 50S subunits. The number of proteins and their electrophoretic mobility seem to remain unaltered during the sporulation process.The ribosomal proteins of a thermosensitive sporulation mutant (ts-4), isolated from stationary phase cultures, under permissive (for sporulation) and non-permissive conditions, did not show any qualitative difference in either of the subunits.The 21S precursor particles derived from log phase cell ribosomes show two different proteins, in addition to those present in the 30 S subunit. It is suggested that these two proteins either disappear or are modified during the maturation process. 相似文献
17.
Ribosomal RNAs are tolerant toward genetic insertions: evolutionary origin of the expansion segments 总被引:1,自引:0,他引:1
Ribosomal RNAs (rRNAs), assisted by ribosomal proteins, form the basic structure of the ribosome, and play critical roles in protein synthesis. Compared to prokaryotic ribosomes, eukaryotic ribosomes contain elongated rRNAs with several expansion segments and larger numbers of ribosomal proteins. To investigate architectural evolution and functional capability of rRNAs, we employed a Tn5 transposon system to develop a systematic genetic insertion of an RNA segment 31 nt in length into Escherichia coli rRNAs. From the plasmid library harboring a single rRNA operon containing random insertions, we isolated surviving clones bearing rRNAs with functional insertions that enabled rescue of the E. coli strain (Δ7rrn) in which all chromosomal rRNA operons were depleted. We identified 51 sites with functional insertions, 16 sites in 16S rRNA and 35 sites in 23S rRNA, revealing the architecture of E. coli rRNAs to be substantially flexible. Most of the insertion sites show clear tendency to coincide with the regions of the expansion segments found in eukaryotic rRNAs, implying that eukaryotic rRNAs evolved from prokaryotic rRNAs suffering genetic insertions and selections. 相似文献
18.
Ankit Gupta Priyanka Shah Afreen Haider Kirti Gupta Mohammad Imran Siddiqi Stuart A. Ralph Saman Habib 《Open biology》2014,4(5)
Apicomplexan protists such as Plasmodium and Toxoplasma contain a mitochondrion and a relic plastid (apicoplast) that are sites of protein translation. Although there is emerging interest in the partitioning and function of translation factors that participate in apicoplast and mitochondrial peptide synthesis, the composition of organellar ribosomes remains to be elucidated. We carried out an analysis of the complement of core ribosomal protein subunits that are encoded by either the parasite organellar or nuclear genomes, accompanied by a survey of ribosome assembly factors for the apicoplast and mitochondrion. A cross-species comparison with other apicomplexan, algal and diatom species revealed compositional differences in apicomplexan organelle ribosomes and identified considerable reduction and divergence with ribosomes of bacteria or characterized organelle ribosomes from other organisms. We assembled structural models of sections of Plasmodium falciparum organellar ribosomes and predicted interactions with translation inhibitory antibiotics. Differences in predicted drug–ribosome interactions with some of the modelled structures suggested specificity of inhibition between the apicoplast and mitochondrion. Our results indicate that Plasmodium and Toxoplasma organellar ribosomes have a unique composition, resulting from the loss of several large and small subunit proteins accompanied by significant sequence and size divergences in parasite orthologues of ribosomal proteins. 相似文献
19.
H G Wittmann 《Proceedings of the Royal Society of London. Series B, Containing papers of a Biological character. Royal Society (Great Britain)》1982,216(1203):117-135
Ribosomes are the only cell organelles occurring in all organisms. E. coli ribosomes, which are the best characterized particles, consist of three RNAs and 53 proteins. All components have been isolated and characterized by chemical, physical and immunological methods. The primary structures of the RNAs and of all the proteins are known. Information about the secondary structure of the proteins derives from circular dichroism measurements and from secondary structure prediction methods. The tertiary structure is being studied by limited proteolysis, proton magnetic resonance and crystallization followed by X-ray analysis. Various methods are being used to elucidate the architecture of the ribosomal particle: three-dimensional image reconstruction of crystals of bacterial ribosomes and/or their subunits; immune electron microscopy; neutron scattering; protein-protein, protein-RNA and RNA-RNA crosslinking; total reconstitution of ribosomal subunits. The results from these studies yield valuable information on the architecture of the ribosomal particle. Many mutants have been isolated in which one or a few ribosomal proteins are altered or even deleted. The genetic and biochemical characterization of these mutants allows conclusions about the importance of these proteins for the function of the ribosome. Ribosomal proteins from various prokaryotic and eukaryotic species have been compared by two-dimensional gel electrophoresis, immunological methods, reconstitution and amino acid sequence analysis. These studies show a strong homology among prokaryotic ribosomal proteins but only a weak homology between proteins from prokaryotic and eukaryotic ribosomes. Comparison of the primary and secondary structures of the ribosomal RNAs from various organisms shows that the secondary structure of the RNA molecules has been strongly conserved throughout evolution. 相似文献
20.
Summary
Bombyx mori L. ribosomal proteins have been analyzed by four related two-dimensional polyacrylamide gel electrophoretic systems (Madjar et al. 1977a). In the small and large subunits are present 32 and 45 proteins, respectively, whose numbering is proposed. No significant differences in composition or migration could be detected between proteins in membranebound ribosomes and free ribosomes. The molecular weights of the proteins vary from 60,000 to less than 10,000. In vivo phosphorylation was investigated by labeling with 32P-orthophosphate. Autoradiograms of four two dimensional gels unambiguously show five labeled ribosomal proteins: S1, S7, L6, L29, and L40. 相似文献