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At least 811 records · Page 45Linked to original sources
PubMed1976

Intraction of aminoacyl-tRNA synthetases with ribosomes and ribosomal subunits.

Salt-washed ribosomes from rabbit reticulocytes stimulate seven partially purified aminoacyl-tRNA synthetases up to threefold: arginyl-, alanyl-, isoleucyl-, lysyl-, methionyl-, phenylalanyl- and valyl-tRNA synthetase. Both isolated subunits but not total ribosomal RNA show the stimulation. The increase in synthetase activity is also found in a highly purified Escherichia coli system employing homogenous (E. coli)phenyl-alanyl-tRNA synthetase and (E. coli)- tRNAPhe. The biological significance of the stimulation is discussed.

Amino Acyl-tRNA Synthetases↗
PubMed1985

tRNA binding to programmed ribosomes increases the ribosomal affinity for tuberactinomycin O.

The binding of 14C-labelled tuberactinomycin O was analysed in equilibrium dialysis cells. The ionic conditions and the concentration of the labelled drug used in the binding assays allowed the binding of just one drug molecule per non-programmed ribosome. Under these conditions, the occupation of the ribosomal P-site by deacylated tRNAPhe in the presence of poly(U) increased the amount of [14C]tuberactinomycin O bound by a factor of two. Kanamycin, gentamicin and neomycin reduced the binding of tuberactinomycin O, whereas chloramphenicol, tetracycline, streptomycin and puromycin had no effect. A stimulation of the binding of tuberactinomycin O was found upon addition of erythromycin.

Anti-Bacterial Agents↗
PubMed1981

Feedback regulation of ribosomal protein synthesis in E. coli: localization of the mRNA target sites for repressor action of ribosomal protein L1.

E. coli ribosomal protein L1 is a translational repressor of the synthesis in vitro of both proteins encoded in the L11 operon (L11 and L1). L1 is shown to act at a single target site within the first 160 bases of the bicistronic mRNA, near (or at) the translation initiation site of the L11 cistron. Synthesis of L1 apparently requires translation of the preceding L11 cistron, allowing regulation of the synthesis of both proteins froma single mRNA target site. This observation suggests a sequential translation mechanism that results in the equimolar synthesis rates of the two proteins observed in vivo. It was found that the presence of 23S rRNA, but not 16S rRNA, relieves translational inhibition by L1. L1 presumably recognizes structural features of the mRNA target site that are homologous to the L1-binding site of 23S rRNA. Although previous work indicated that translationally inhibited ribosomal protein mRNA is degraded in vivo, L1 repressor action in the present in vitro system was found not to involve mRNA degradation.

Bacterial Proteins↗
PubMed1987

Increases in ribosomal RNA within the denervated neuropil of the dentate gyrus during reinnervation: evaluation by in situ hybridization using DNA probes complementary to ribosomal RNA.

Previous studies have revealed that there are increases in the incorporation of [3H]amino acids into protein in the denervated neuropil of the dentate gyrus during periods of reactive synaptogenesis. The present study evaluates whether the increase in incorporation reflects an increase in protein synthetic machinery (ribosomes) in the denervated zone. We evaluated the distribution of ribosomal RNA (rRNA) in the denervated dentate gyrus 2-14 days after unilateral destruction of the entorhinal cortex using DNA probes complementary to rRNA for in situ hybridization. Animals with comparable lesions were injected with [3H]leucine 30 min prior to sacrifice and prepared for autoradiography in order to define the extent of protein synthesis within the denervated neuropil. Quantitative analyses revealed that the increases in [3H]leucine incorporation were accompanied by increases in labeling with the rRNA probe. In both cases, the increases were first apparent at 2 days postlesion, reached a peak on day 6, and then declined between 8 and 14 days postlesion. Plots of grain density across the neuropil revealed that the increases in rRNA, like the increases in amino acid incorporation, occurred selectively within the denervated portion of the neuropil. We propose that increased incorporation of protein precursor is the result of an increase in protein synthetic machinery within the denervated neuropil. These increases may reflect in part the increases that we have previously noted in polyribosomes under dendritic spines.

Animals↗
PubMedDate not supplied

The primary structure of rat ribosomal proteins P0, P1, and P2 and a proposal for a uniform nomenclature for mammalian and yeast ribosomal proteins.

The covalent structures of rat ribosomal proteins P0, P1, and P2 were deduced from the sequences of nucleotides in recombinant cDNAs. P0 contains 316 amino acids and has a molecular weight of 34,178; P1 has 114 residues and a molecular weight of 11,490: and P2 has 115 amino acids and a molecular weight of 11,684. The rat P-proteins have a near identical (16 of 17 residues) sequence of amino acids at their carboxyl termini and are related to analogous proteins in other eukaryotic species. A proposal is made for a uniform nomenclature for rat and yeast ribosomal proteins.

Amino Acid Sequence↗
PubMed1981

Polyamine concentration, activity of the ribosomal dissociation factor and proportion of ribosomes active in protein synthesis in the 4-dimethylaminoazobenzene-induced rat hepatoma.

4-Dimethylaminoazobenzene (DAB) induced liver tumour tissue showed a reduced proportion of ribosomes active in protein synthesis compared with control liver. Tumour cell extracts caused an increased association of ribosome subunits into inactive 80 S monomers, compared with the dissociation into active subunits caused by normal liver extracts. These findings may be explained, at least in part, by the increased proportion of spermidine to putrescine found in tumour tissue which would predispose towards association of the subunits.

Animals↗
PubMed1982

Size heterogeneity of ribosomal RNA in eukaryote evolution--1. rRNA molecular weights in species containing intact large ribosomal subunit RNA.

1. The molecular weights of the two major rRNA species (L-rRNA, large ribosomal subunit RNA, and S-rRNA, small ribosomal subunit RNA) of a variety of deuterostomia, green plants and fungi have been investigated by gel electrophoresis in 99% formamide, pH 9; the overall pattern obtained under these conditions differs to some extent from that deduced by electrophoresis in neutral-salt solutions. 2. The molecular weights of the deuterostomian S-rRNA species have been conserved at a value of 0.65 X 10(6), whereas those of the L-rRNA have been kept at 1.40 X 10(6) in the lower species but have increased to 1.55 X 10(6) in birds and to 1.65 X 10(6) in mammals. 3. The molecular weights of the L-rRNA and S-rRNA components of the green plants (Dycotyledons, Monocotyledons and Gymnosperms) have been generally conserved at 1.30 X 10(6) and 0.65 X 10(6). 4. The molecular weight of the L-rRNA of the fungi has been conserved at 1.36-1.38 X 10(6), being 0.1 X 10(6) daltons heavier than that of the plants; the S-rRNA exhibits a limited degree of variability, ranging between 0.65 X 10(6) and 0.72 X 10(6).

Animals↗
PubMed1984

Ribosomal RNA genes of Trypanosoma brucei: mapping the regions specifying the six small ribosomal RNAs.

There are six small ribosomal RNAs in trypanosome ribosomes. sRNA3 and sRNA5 of Trypanosoma brucei brucei have been partially sequenced. Sequence homologies indicate that sRNA3 is 5.8S RNA and sRNA5 is 5S RNA of T. b. brucei. The regions specifying these two, and the remaining four small RNAs, have been identified within clones of rRNA genes and in the genome. Five of the small RNAs, 1, 2, 3, 4 and 6, hybridise exclusively within the major rRNA gene repeat. A map of the regions specifying these small RNAs is presented. sRNA3 (5.8S RNA) hybridises to a region corresponding to the transcribed spacer of other eukaryotes. sRNA1 hybridises to a region between sequences specifying the two large subunit RNA molecules of 2.3 kb and 1.8 kb. Sequences specifying sRNAs 2 and 4 are present near the sequence specifying sRNA1, while sRNA6 appears to be specified 3' to the sequence specifying the 1.8-kb RNA sequence. In addition regions of secondary hybridisation for small RNAs 2, 3, 4 and 6 have also been identified. Though sRNA5 (5S RNA) hybridises within the major rRNA repeat, a separate 5S RNA gene repeat with unit size of 760 bp is also present. It is 10 to 20 times more abundant than the major rRNA gene repeat.

Animals↗
PubMed1986

Characterisation of an mRNA encoding a human ribosomal protein homologous to the yeast L44 ribosomal protein.

We describe the isolation and characterisation of a full-length cDNA sequence (pZH-21) of a human ribosomal protein (rp) mRNA isolated from a cDNA library constructed from the human ZR-75-1 mammary tumour cell-line. The predicted protein is highly basic and shows 72% homology at the amino acid (aa) level with yeast rp L44. Comparative RNA blotting of ZR-75-1 poly(A)+ RNA isolated from cells cultured in the presence of the anti-oestrogen tamoxifen demonstrates the presence of a number of mRNA species whose concentration is elevated co-ordinately 5-6-fold in the presence of 17beta-oestradiol. Insulin in the presence of tamoxifen, also enhanced rp mRNA levels suggesting increased levels are a reflection of cell proliferation as opposed to specific hormonal regulation. Genomic analysis demonstrates the presence of a family of related human sequences, and homology with rat and guinea pig rp genes, but not yeast DNA. The conservation of rp aa sequence, in the absence of detectable homology at the nucleotide (nt) level, points to an important common functional role of the L44 protein in ribosome structure and function in man and yeast.

Amino Acid Sequence↗
PubMed2006

Preparation of ribosomes loaded with truncated nascent proteins to study ribosome binding to mammalian mitochondria.

Supporting a co-translational model of protein import into mitochondria, we have previously shown that ribosome-nascent chain complexes (RNCs) specifically bind to mitochondria. When producing RNCs using the rabbit reticulocyte lysate in vitro translation system, it was necessary to maximize ribosome loading with truncated nascent proteins because it had a direct impact on RNC binding. We describe here the optimal conditions for preparing RNCs. We show that translation temperature and reaction time are two critical factors, with 30 degrees Celsius and 15min being optimal, respectively. We also show that transcription reactions can be used directly in the translation reaction to create RNCs.

Animals↗
PubMed2006

Isolation and characterization of mitochondrial ribosomes and ribosomal subunits from Leishmania tarentolae.

We have analyzed Leishmania tarentolae mitochondrial ribonucleoprotein (RNP) complexes using the 9S small subunit (SSU) rRNA and the 12S large subunit (LSU) rRNA as markers, and have identified a 50S RNP particle as the putative mitochondrial monosome, a 40S particle as the putative LSU and a 30S particle as the putative SSU. These assignments are supported by morphological analysis by cryo-electron microscopy and proteomics analyses by mass spectrometry. The presence of additional rRNA-containing particles complicated the analysis and most likely was the basis for previous difficulties in identification of these ribosomes; thus, in addition to the monosomes and their subunits, there are abundant stable 45S particles (SSU(*)) containing only 9S rRNA, which may represent homodimers of the SSU or SSU associated with additional proteins, and variable minor amounts of 65S and 70S particles, which represent homodimers of the LSU and SSU(*), respectively. These additional rRNA particles might be due to the lengthy mitochondrial isolation and ribosome isolation procedures or may be present in vivo and play yet undetermined roles.

Animals↗
PubMed1980

Protection against non-enzymatic deacylation of Met-tRNA Met m by the ribosome : an approach to the measurement of aminoacyl-tRNA : ribosome affinity constants.

When bound to E. coli 70S ribosomes in the presence of poly (A, U, G) at 37 degrees C, Met-tRNA Met m is hydrolysed 20 times slower than when free in solution under the same conditions (lifetimes of 8.3 h and 22 min respectively). It is shown how this large difference in rate of hydrolysis may be used to study the equilibrium of aminoacyl-tRNA binding to ribosomes.

Escherichia coli↗
PubMed1983

Comparison of ribosomal entry and acceptor transfer ribonucleic acid binding sites on Escherichia coli 70S ribosomes. Fluorescence energy transfer measurements from Phe-tRNAPhe to the 3' end of 16S ribonucleic acid.

Distances were measured by nonradiative energy transfer from fluorescent probes specifically located on one of three points of yeast or Escherichia coli Phe-tRNAPhe enzymatically bound to the entry site or to the acceptor site of E. coli 70S ribosomes to energy-accepting probes on the 3' end of the 16S ribonucleic acid (RNA) of the 30S subunit. The Y base in the anticodon loop of yeast tRNAPhe was replaced by proflavin. Fluorescein isothiocyanate was attached to the X base (position 47) of E. coli tRNAPhe. E. coli tRNAPhe which had been photochemically cross-linked between positions 8 and 13 followed by chemical reduction to form a fluorescent probe was also used. Labeled tRNAs were aminoacylated and enzymatically bound to the ribosome in the presence of elongation factor Tu and guanosine 5'-triphosphate (acceptor-site binding) or a nonhydrolyzable analogue (entry-site binding). Nonradiative energy transfer measurements were made of the distances between fluorophores located on the Phe-tRNA and the fluorophore at the 3' end of 16S RNA. Calculations were based on comparison of the fluorescence lifetime of the energy donor, located on the Phe-tRNA, in the absence and presence of an energy acceptor on the 3' end of the 16S RNA. Under both sets of binding conditions, the distances to the 3' end of 16S RNA were found to be the following: cross-linked tRNA, greater than 69 A; Y base of tRNA, greater than 61 A. The distance between the 3' end of 16S RNA and the X base of tRNA was found to be 81 A under acceptor-site binding conditions but greater than 86 A under entry-site binding conditions.

Energy Transfer↗
PubMed1981

Position of transfer ribonucleic acid on Escherichia coli ribosomes. Distance from the 3' end of 16S ribonucleic acid to three points on phenylalanine-accepting transfer ribonucleic acid in the donor site of 70S ribosomes.

Escherichia coli 16S RNA from 30S ribosomal subunits was isolated, oxidized at the 3' end, and labeled with the thiosemicarbazide derivatives of fluorescein or eosin. Labeled 16S RNA was reconstituted into 30S subunits. They were almost fully active compared to 30S subunits reconstituted from unlabeled 16S RNA by using a poly(uridylic acid)-directed polyphenylalanine synthesis assay. Fluorophores were placed at three different positions of tRNAPhe. E. coli and yeast tRNAPhe were oxidized at the 3' end and labeled with the thiosemicarbazide derivative of fluorescein or with the hydrazide of N-methylanthranilic acid. The Y base in the anticodon loop of yeast tRNAPhe was replaced by proflavin or 1-aminoanthracene. Also, E. coli tRNAPhe was photochemically cross-linked between 4-thiouridine at position 8 and cytidine at position 13. After reduction, this site was used as a fluorescent probe. The labeled tRNAs were bound into the peptidyl site of 70S ribosomes, and then the distances from the fluorophore in the modified tRNA to the fluorophore at the 3' end of 16S RNA were measured by nonradiative energy transfer. Calculations were based on measurements of fluorescence lifetimes. The distances to the 3' end of 16S RNA were found to be as follows: 3' end of tRNA, 67-74 A; cross-linked t RNA, 53-60 A; anticodon loop of tRNA, greater than 61 A.

Binding Sites↗
PubMed1982

Independent binding sites in mouse 5.8S ribosomal ribonucleic acid for 28S ribosomal ribonucleic acid.

Limited digestion of mouse 5.8S ribosomal RNA (rRNA) with RNase T2 generates 5'- and 3'-terminal "half-molecules". These fragments are capable of independently and specifically binding to 28S rRNA, so there exist at least two contacts in the 5.8S rRNA for the 28S rRNA. The dissociation constants for the 5.8S/28S, 5' 5.8S fragment/28S, and 3' 5.8S fragment/28S complexes are 9 x 10(-8) M, 6 x 10(-8) M, and 13 x 10(-8) M, respectively. Thus, each of the fragment binding sites contributes about equally to the overall binding energy of the 5.8S/28S rRNA complex, and the binding sites act independently, rather than cooperatively. The dissociation constants suggest that the 5.8S rRNA termini from short, irregular helices with 28S rRNA. Thermal denaturation data on complexes containing 28S rRNA and each of the half-molecules of 5.8S rRNA indicate that the 5'-terminal binding site(s) exist(s) in a single conformation while the 3'-terminal site exhibits two conformational alternatives. The functional significance of the different conformational states is presently indeterminate, but the possibility they may represent alternative forms of a conformational switch operative during ribosome function is discussed.

Animals↗
PubMed1975

Methylation of the ribosomal proteins in Escherichia coli. Nature and stoichiometry of the methylated amino acids in 50S ribosomal proteins.

Methylated ribosomal proteins from Escherichia coli 50S subunit are localized by growing cells in a medium containing (1-14C)methionine and (3H-methyl)-methionine and comparing the 3H/14C ratio for each of the 50S ribosomal proteins. The following proteins are methylated: L11, L1, L3, L5, L7, L8, L9, L12, L18, and L33. The nature and stoichiometry of the methylated amino acid(s) in each of the methylated proteins are determined. Protein L11 is the most heavily methylated of all the 50S subunit proteins. This protein has previously been implicated in the peptidyl transferase reaction during protein synthesis (K. H. Nierhaus and V. Montejo (1973), Proc. Nat. Acad. Sci. U. S. 47, 1588-1602). Three proteins (L1, L3, and L5) have intermediate levels of methylation and contain about 0.4-0.6 methyl groups each per molecule of protein. Five other proteins (L7, L8, L9, L12, and L18) are also methylated to a slight extent (-0.1 methyl group/molecule of protein). One unknown methylated neutral amino acid was detected in protein L11 and at least one and possibly two other unidentified methylated amino acids appeared to be present in protein L33.

Amino Acids↗
PubMed1971

The arrangement of ribosomes in ribosome tetramers from hypothermic chick embryos.

1. Ribosomes and the tetramer arrangement peculiar to the tissues of chick embryos exposed to low temperatures were separated by sucrose-density-gradient centrifugation, and the effects of variation of the concentrations of Mg(2+), Ca(2+) and K(+) studied. 2. Lowering of the Mg(2+) concentration from standard buffer conditions caused a reversible dissociation of tetramers into monomers and of these into subunits. 3. Ca(2+) replaced Mg(2+) in causing the re-formation of tetramers and monomers from subunits after dissociation in low Mg(2+) concentrations. 4. Ca(2+) also caused an almost complete conversion of monomers into dimers in the presence of Mg(2+). 5. The effect of Ca(2+) on the formation of dimers was abolished by pretreatment of the ribosomes with ribonuclease, but the re-formation of tetramers was unaffected. 6. Increase of the K(+) concentration from that of the standard buffer caused dissociation of monomers and dimers into subunits. 7. Raised K(+) concentration also caused a stepwise alteration of the tetramer from a particle with a sedimentation coefficient of 197S, which constitutes the bulk of the tetramer at low K(+) concentrations, first to a 184S peak and finally to material with a sedimentation coefficient of about 155S. 8. The implications of these results on hypotheses of the arrangement of the individual monomers in the tetramer are discussed and a new model for the structure is proposed.

Animals↗