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At least 937 records · Page 52Linked to original sources
PubMed1976

Arthropod ribosomes. Integrity of ribosomal ribonucleic acids from aphids and water fleas.

The 28 S aphid rRNA differs from those of the other insects in two points: (1) it lacks the primary nick; (2) it is larger by 0.2 - 106 daltons. The 28 S rRNA from water fleas also, like that from crayfish, is larger than those from comon insects by 0.2 - 106 daltons. These crustacean 28-S rRNAs were shown to contain the primary nick, which is probably not located in the central point. The 18-S rRNAs from aphids and water fleas had molecular weights significantly larger than 0.7 - 106, the common vaalue for the eukaryotic 18 S rRNA. It was was suggested that the 3'-terminal base sequences of these RNAs are different from the common sequences of these RNAs are different from the common sequence proposed for the 18 S rRNAA of eukaryotes. These exceptional characteristics of the rRNAs from the parthenogenetic animals may provide a probe for general functions of the rRNA in the eukaryotic ribosomes.

Animals↗
PubMed1988

Crosslinking of ribosomal protein S18 to 16 S RNA in E.coli ribosomal 30 S subunits by the use of a reversible crosslinking agent: trans-diamminedichloroplatinum(II).

We have previously developed [(1987) Biochemistry 26, 5200-5208] the use of trans-diamminedichloroplatinum(II) to induce reversible RNA-protein crosslinks in the ribosomal 30 S subunit. Protein S18 and, to a lesser extent, proteins S13/S14, S11, S4 and S3 could be crosslinked to the 16 S rRNA. The aim of the present work was to identify the crosslinking sites of protein S18. Three sites could be detected: a major one located in region 825-858, and two others located in regions 434-500 and 233-297. This result is discussed in the light of current knowledge of the topographical localization of S18 in the 30 S subunit and of its relation with function.

Base Sequence↗
PubMed1985

A mutation in an Escherichia coli ribosomal RNA operon that blocks the production of precursor 23 S ribosomal RNA by RNase III in vivo and in vitro.

We have isolated on a multicopy plasmid a mutant rrnB ribosomal RNA operon containing a 130 base-pair deletion immediately preceding the 23 S rRNA gene. The deletion shortens by just three base-pairs the 26 base-pair complementarity of the sequences that flank the 23 S rRNA gene, and which normally form an RNase III cleavage site in the rrnB primary transcript. Both in vivo and in vitro, cleavage at the altered RNase III site was almost completely abolished by the mutation. Our results therefore indicate that even a small perturbation of the double-stranded region normally recognized by RNase III strongly inhibits the action of the enzyme.

Autoradiography↗
PubMed1976

Synthesis of ribosomal RNA in E. coli: analysis using deletion mutants of a lambda transducing phage carrying ribosomal RNA genes.

Transducing phage lambdarifd18 carries on rRNA transcription unit containing genes for 5S, 16S, and 23S rNAs and also tRNAGlu/2. Mutants were isolated from this phage that carry deletions removing various amounts of the distal end of this transcription unit. These deletions were physically mapped on the lambdarifd18 phage genome. Synthesis of rRNAs and of tRNAGlu/2 was examined in ultraviolet-irradiated E. coli cells infected with lambdarifd18 or with various deletion mutants. It was observed that mutant phages in which the distal end (the 5S rRNA gene and a part of the 23S rRNA gene) of the rRNA transcription unit is deleted can still synthesize both 16S rRNA (or its precursor) and tRNAGlu/2. Apparently, the post-transcriptional cleavage that produces these RNA molecules does not require the presence of the entire transcription unit, that is, it can take place without the complete structure of the transcript ("30S pre-ribosomal RNA"). In addition, the experimental results support the gene order, 16S rRNAGlu/2, 23S rRNA, and 5S rRNA genes, in the rRNA transcription unit carried by lambdarifd18.

Chromosome Mapping↗
PubMed1982

Size heterogeneity of ribosomal RNA in eukaryote evolution--2. rRNA molecular weights in species containing discontinuous large ribosomal subunit RNA.

1. The molecular weights and the integrity of the principal rRNA species derived from the large and small ribosomal subunit (respectively, L-rRNA and S-rRNA) of several species of Protostomia and Protozoa have been investigated. 2. Using gel electrophoresis in formamide, the molecular weights of protostomian L-rRNA species have been found to range from 1.30 X 10(6) (Annelida) to 1.61 X 10(6) (Diptera); those of the S-rRNA's cover the range 0.65 X 10(6) (Annelida)-0.81 X 10(6) (Diptera). 3. Both rRNA components have incurred extensive changes among the Protozoa; the L-rRNA ranges in weight from 1.35 X 10(6) (T. pyriformis) to 1.57 X 10(6) (A. castellanii) and the S-rRNA from 0.70 X 10(6) of T. pyriformis to 0.85 X 10(6) of A. castellanii and E. gracilis. 4. The L-rRNA components of all the species investigated are discontinuous molecules endowed with a latent median break; depending on whether the nick is located at the centre of the L-rRNA chain, or lies off-centre, the molecular weight of the S-rRNA equals that of either both, or only one, of the two fragments composing the L-rRNA.

Animals↗
PubMed2005

Ribosome builder: a software project to simulate the ribosome.

The Ribosome Builder is a software project that provides tools and techniques to create dynamic models of macromolecular systems from the rapidly growing numbers of atomic structural models. It includes a computer program that allows the user to assemble the multiple molecular components within a 3D space and to define the hypothetical interactions of these components with the initial goal of understanding protein translation at an atomic level of detail. The program employs a simplified molecular dynamics forcefield that can simulate the long time-scale events, such as docking of translation factors and mRNA translocation. An embedded scripting language and Application Programming Interface (API) enable the creation of Steered Molecular Dynamics (SMD) simulations through the programmable application of external forces and torques on atoms and bonds. A graphical interface is provided for displaying and interacting with models, recording movies of molecular dynamics movements, and creating annotated 3D simulations of complex macromolecular events. Initial applications of the project include simulation of tetraloop folding, docking of an mRNA on the 30S subunit and a schematic simulation of the translation elongation cycle. The program is an open source project released under the GNU public license.

Computer Simulation↗
PubMed1999

Determination of the nucleotide sequence of the 23S ribosomal RNA and flanking spacers of an Enterococcus faecium strain, reveals insertion-deletion events in the ribosomal spacer 1 of enterococci.

The usefulness of 16S-23S (ITS1) and 23S-5S (ITS2) ribosomal spacer nucleotide sequence determination, as a complementary approach to the biochemical tests traditionally used for enterococcal species identification, is shown by its application to the identification of a strain, E27, isolated from a natural bacteria mixture used for cheese production. Using combined approaches we showed, unambiguously, that strain E27 belongs to the Enterococcus faecium species. However, its ITS1 region has an interesting peculiarity. In our previous study of ITS1s from various enterococcal species (NAIMI et al., 1997, Microbiology 143, 823-834), the ITS1s of the two E. faecium strains studied, were found to contain an additional 115-nt long stem-loop structure as compared to the ITS1s of other enterococci, only one out of the 3 ITS1s of E. hirae ATCC 9790, was found to contain a similar 107-nt long stem-loop structure. The ITS1 of strain E27 is 100% identical to that of E. faecium ATCC 19434T, except that the 115-nt additional fragment is absent. This strongly suggests the existence of lateral DNA transfer or DNA recombination events at a hot spot position of the ITS1s from E. faecium and E. hirae. Small and large ITS1 nucleotide sequence determination for strain E27 generalized the notion of two kinds of ITSs in enterococci: one with a tRNA(Ala) gene, one without tRNA gene. To complete strain E27 characterization, its 23S rRNA sequence was established. This is the first complete 23S rRNA nucleotide sequence determined for an enterococcal species.

Base Sequence↗
PubMed1981

Effect of point mutations on 5.8S ribosomal ribonucleic acid secondary structure and the 5.8S--28S ribosomal ribonucleic acid junction.

Naturally occurring differences in the nucleotide sequences of 5.8S ribosomal ribonucleic acids (rRNAs) from a variety of organisms have been used to study the role of specific nucleotides in the secondary structure and intermolecular interactions of this RNA. Significant differences in the electrophoretic mobilities of free 5.8S RNAs and the thermal stabilities of 5.8S--28S rRNA complexes were observed even in such closely related sequences as those of man, rat, turtle, and chicken. A single base transition from a guanylic acid residue in position 2 in mammalian 5.8S rRNA to an adenylic acid residue in turtle and chicken 5.8S rRNA results both in a more open molecular conformation and in a 5.8S--28S rRNA junction which is 3.5 degrees C more stable to thermal denaturation. Other changes such as the deletion of single nucleotides from either the 5' or the 3' terminals have no detectable effect on these features. The results support secondary structure models for free 5.8S rRNA in which the termini interact to various degrees and 5.8S--28S rRNA junctions in which both termini of the 5.8S molecule interact with the cognate high molecular weight RNA component.

Alkaline Phosphatase↗
PubMed1976

Role of ribosomal RNA methylases in the regulation of ribosome production in mammalian cells.

The activity of rRNA methylases was stimulated by high-energy precursors of RNA (ribonucleoside triphosphates) and inhibited by degradation products of RNA (ribonucleotides and oligoribonucleotides). The response of methylases from rat Novikoff ascites tumor and liver to these metabolites was strikingly different. The highly active tumor enzymes responded preferentially to inhibition by catabolic metabolites, whereas the less active liver enzymes responded exclusively to stimulation by anabolic metabolites. When the activity of rRNA methylases was assayed in response to increasing concentration of S-adenosylmethionine, the tumor enzymes responded with a hyperbolic substrate dependence curve and the liver enzymes with a sigmoidal curve. In the presence of an inhibitory dinucleotide, ApA, the tumor enzymes responded with a sigmoidal curve; in the presence of a stimulator, adenosine 5'-triphosphate, the liver enzymes responded with a hyperbolic substrate concentration curve. When normal rats were subject to a series of treatments by thioacetamide, a hepatocarcinogen, the liver nucleolar rRNA methylases became responsive to inhibition by ApA and relatively unresponsive to stimulation by adenosine 5'-triphosphate. When tumor-bearing rats were treated with polyinosinate:polycytidylate, an antitumor agent, the tumor nucleolar rRNA methylases became unresponsive to inhibition by ApA and more responsive to stimulation by adenosine 5'-triphosphate. A correlation was noted between increased methylation efficiency in vivo and increased stability of nucleolar RNA during incubation in vitro, or vice versa. These results are interpreted to indicate that rRNAmethylases are regulated by cellular metabolites during the nucleolar biosynthesis of ribosomes and that rRNA methylases may provide a favorable site for selective action by cancer chemotherapeutic agents.

Adenosine Triphosphate↗
PubMed2004

Fluorescence-based approach for detecting and characterizing antibiotic-induced conformational changes in ribosomal RNA: comparing aminoglycoside binding to prokaryotic and eukaryotic ribosomal RNA sequences.

Aminoglycoside antibiotics bind specifically to a conserved sequence of the 16S ribosomal RNA (rRNA) A site and interfere with protein synthesis. One model for the mechanism underlying the deleterious effects of aminoglycosides on protein synthesis invokes a drug-induced conformational change in the rRNA that involves the destacking of two adenine residues (A1492 and A1493 in Escherichia coli) at the A site. We describe here a fluorescence-based approach for detecting and characterizing this drug-induced conformational change in the target rRNA. In this approach, we insert the fluorescent base analogue 2-aminopurine in place of A1492 in an E. coli 16S rRNA A-site model oligonucleotide (EcWT) as well as in a mutant form of this oligomer (A1408G) in which A1408 has been replaced with a guanine. The presence of guanine at 1408 instead of adenine represents one of the major sequence differences between prokaryotic and eukaryotic A sites, with the latter A sites being resistant to the deleterious effects of aminoglycosides. Binding of the aminoglycoside paromomycin to the 2AP-substituted forms of EcWT and A1408G induced changes in fluorescence quantum yield consistent with drug-induced base destacking in EcWT but not A1408G. Isothermal titration calorimetry studies reveal that paromomycin binds to the EcWT duplex with a 31-fold higher affinity than the A1408G duplex, with this differential affinity being enthalpic in origin. In the aggregate, these observations are consistent with both rRNA binding affinity and drug-induced base destacking being important determinants in the prokaryotic specificity of aminoglycosides. Combining fluorescence quantum yield and lifetime data allows for quantification of the extent of drug-induced base destacking, thereby providing a convenient tool for evaluating the relative impacts of both novel and existing A-site targeting ligands on rRNA conformation and potentially for predicting relative antibiotic activities and specificities.

2-Aminopurine↗
PubMed1999

Ribosomal RNAs synthesized by isolated squid nerves and ganglia differ from native ribosomal RNAs.

The large rRNA of the squid comprises two chains that may be dissociated by heating at 65 degrees C. A single chain constitutes the small rRNA. Surprisingly, the RNAs synthesized by dissected squid fin nerves and stellate nerves and ganglia differed in size from native rRNAs and did not manifest thermal instability. Nonetheless, they resembled native rRNAs in relative abundance, subcellular distribution, lack of poly(A), and metabolic stability. In addition, newly synthesized RNA was localized in nerve and glial cells, as shown by autoradiographic analysis, and was assembled into 80S ribosomes, which supported the synthesis of neuron-specific neurofilament proteins. Following incubation of nerves and ganglia for >10 h, native rRNAs started to disappear, while two major newly synthesized RNAs progressively accumulated. As a result, after 20 h, native rRNAs were substituted by the two novel RNAs. With use of 32P-cDNA synthesized from the latter RNAs as a probe, the novel RNAs demonstrated a considerable degree of homology with native rRNA in northern analysis. Taken together, the data suggest that in dissected squid nerves and ganglia, the synthesis of native rRNAs is gradually terminated while two novel rRNAs are being synthesized, presumably as a correlate of reactive gliosis and/or neuronal degeneration/regeneration.

Animals↗
PubMed1970

Ribosomal proteins. 8. Molecular weights of isolated ribosomal proteins of Escherichia coli.

Molecular weights of the isolated proteins from the 30S and 50S ribosomal subunits of Escherichia coli were determined by two independent methods: polyacrylamide gel electrophoresis with sodium dodecylsulphate and equilibrium sedimentation. The values for the molecular weights determined by gel electrophoresis range from 10,900 to 65,000 for the proteins of the 30S subunit and from 9,600 to 31,500 for those of the 50S subunit, with number averages of 19,000 and 16,300, respectively, in agreement with those obtained by equilibrium sedimentation.

Bacterial Proteins↗
PubMed1972

Early estrogen action: stimulation of the metabolism of high molecular weight and ribosomal RNAs (estradiol-17 -RNA isolation-uterus-ribosomal RNA precursors-actinomycin D).

Samples of RNA, isolated from uteri of ovariectomized adult rats treated with estrogen, have been analyzed on sucrose gradients. Treatment with estrogen either for 20 min or 2 hr increased the specific activity of all classes of uterine RNA, but produced no significant alteration in the distribution of radioactivity in the gradients, when animals received [(3)H]uridine intraperitoneally 15 min before they were killed. After labeling periods of 30 min, 1 hr, or 2 hr, however, the RNAs isolated from animals treated with estrogen had a smaller percentage of rapidly sedimenting (faster than 28S) species of RNA than did RNA from animals not treated with the hormone. The decreased percentage of high molecular weight RNA correlated with increases in both the specific activity of 28S and 18S RNA and the concentration of RNA in the whole organ. The labeled RNA of high molecular weight was also demonstrated, by the use of actinomycin D in vivo, to have a more rapid turnover rate in the estrogen-stimulated uterus. Our results indicate that estrogen increases not only the rate of synthesis of ribosomal RNA in the uterus of the ovariectomized adult rat, but also the rate or efficiency of processing of precursor RNA species of high molecular weight.

Animals↗
PubMed1974

Replacement of ribosomal protein S1 by interference factor ialpha in ribosomal binding of phage Ms2 RNA.

The MS2 RNA binding capacity of 30S ribosomal subunits, which is lost when protein S1 is removed, can be restored following incubation with interference factor ialpha and repelleting. Polyacrylamide-agarose gel electrophoresis shows that, under these conditions, a faster moving, non-RNA binding 30S species, which contains no S1, is converted to a slower moving RNA-binding one, having the same mobility as the 30S species that contains protein S1. Factor ialpha binds to single-stranded RNAs in a pattern that closely resembles the RNA binding pattern of initiation factor IF-3.

Bacterial Proteins↗
PubMed1988

Circular ribosomal DNA plasmids transform Tetrahymena thermophila by homologous recombination with endogenous macronuclear ribosomal DNA.

We transformed the ciliate Tetrahymena thermophila by microinjection of circular plasmids containing the ribosomal RNA gene (rDNA). In the somatic macronucleus of Tetrahymena, the rDNA is in the form of linear palindromic molecules. The rDNA molecules from the C3 strain have a replication advantage over rDNA from both B strain and the C3 rDNA mutant rmm1. We constructed two circular plasmids carrying replication origin sequences from C3 rDNA and a point mutation (Pmr) in the 17S rRNA gene that confers resistance to the antibiotic paromomycin. One plasmid contained a single complete copy of the rRNA gene and its flanking sequences, while the other had an additional rDNA origin of replication. In all B or rmm1 Tetrahymena cell lines transformed with the plasmids, rDNA sequences from the plasmid were found in palindromic rDNA molecules. In one transformant line, a small amount of the plasmid was also retained in a form with the original circular restriction map. Our results show that the plasmids underwent homologous recombination with one arm of the endogenous rDNA to give heteropalindromic rDNA, or with both arms of the palindrome to form homopalindromic rDNA. The resulting recombinant molecules were able to replace the recipient's original rDNA completely, providing strong evidence that C3 rDNA sequences in the donor DNAs confer a replication advantage over recipient rDNA. Thus microinjection of circular plasmids provides a method for replacement of an endogenous gene or gene fragment with exogenous sequences.

Animals↗
PubMed1987

The ribosomal E site at low Mg2+: coordinate inactivation of ribosomal functions at Mg2+ concentrations below 10 mM and its prevention by polyamines.

Under standard conditions (Mg2+/150 mM NH4+) ribosomes can quantitatively participate in tRNA binding at Mg2+ concentrations of 12 to 15 mM. The overall poly(U)-directed Phe incorporation and the extent of tRNA binding to either P, E or A sites decrease in a parallel manner when the Mg2+ concentration is lowered below 10 mM. At 4 mM the inactivation amounts to about 80%. The coordinate inactivation of all three binding sites is accompanied by an increasing impairment of the ability to translocate A-site bound AcPhe-tRNA to the P site. The translocation efficiency is already reduced at 10 mM Mg2+, and is completely blocked at 6-8 mM. The severe inactivation seen at 6 mM Mg2+ vanishes when the polyamines spermine (0.6 mM) and spermidine (0.4 mM) are present in the assay; tRNA binding again becomes quantitative, the total Phe synthesis even exceeds that observed in the absence of polyamines by a factor of 4. In the presence of polyamines and low Mg2+ (3 and 6 mM) two essential features of the allosteric three-site model (Rheinberger and Nierhaus, J. Biol. Chem. 261, 9133 (1986] are demonstrated. 1) Deacylated tRNA is not released from the P site, but moves to the E site during the course of translocation. 2) Occupation of the E site reduces the A site affinity and vice versa (allosteric interactions between E and A sites). The quality of an in vitro system for protein synthesis can be assessed by two criteria. First, the incubation conditions must allow a near quantitative tRNA binding. Secondly, protein synthesis should proceed with near in vivo rate and accuracy. The 3 mM Mg2+/NH4+/polyamine-system seems to be the best compromise at present between these two requirements.

Allosteric Regulation↗