Magnesium ions and the structure of Escherichia coli ribosomal ribonucleic acid.
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ABSTRACT:
SUBMITTER: Rodgers A
PROVIDER: S-EPMC1265099 | biostudies-other | 1966 Jul
REPOSITORIES: biostudies-other
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Project description:1. RNA synthesized by Escherichia coli during one-hundredth of the generation time contains two fractions distinguishable by hybridization with homologous DNA. One fraction, approximately 30% of the newly synthesized RNA, did not compete with ribosomal RNA, being apparently messenger RNA. The other fraction, approximately 70% of the newly made RNA, hybridized as ribosomal RNA. These values are comparable with previous estimates (McCarthy & Bolton, 1964; Pigott & Midgley, 1968). 2. Hybridization-competition experiments showed that the newly made RNA associated with 70s ribosomes and larger ribosome aggregates was a mixture of ribosomal RNA and messenger RNA, whereas that associated with nascent ribosomal subunits consisted exclusively of ribosomal RNA. This observation provides means by which newly synthesized ribosomal RNA can be isolated free from messenger RNA. 3. Newly made ribosomal RNA in nascent ribosomal subunits was sensitive to shear under conditions where ribosomal RNA in mature ribosomes was shear-resistant. Thus, when RNA was extracted from cells of E. coli disrupted by mechanical means, newly made ribosomal RNA appeared heterogeneous in size, sedimenting as a broad peak extending from 8s to 16s. 4. Newly synthesized ribosomal RNA in nascent ribosomal subunits was rapidly degraded in the presence of actinomycin D and during glucose starvation. 5. Newly synthesized ribosomal RNA stimulated amino acid incorporation in a system synthesizing protein in vitro to the same extent as the RNA which contained the messenger RNA fraction.
| S-EPMC1185102 | biostudies-other
Project description:1. The u.v.-absorption spectrum of ribosomal RNA from rabbit reticulocytes was studied as a function of temperature at different pH values. The changes in the spectrum over the range 220-320mmu were interpreted on the basis of the assumption that the effect of denaturation and ionization are additive. The results suggest that in neutral salt solutions the secondary structure of the ribosomal RNA samples studied is due to two species of helical segments stabilized principally, if not solely, by complementary base pairs but differing in nucleotide composition: each species appears to be heterogeneous in other respects in view of the breadth of the melting ranges. 2. The number of base pairs per helical segment was estimated to be small (between 4 and 17) on the basis of the relation between melting temperature and chain length previously established by Lipsett and others for model compounds. Small fragments (about 2s) obtained by alkaline hydrolysis appeared to form the same helical segments as the intact molecule in accord with the estimated size of these segments. 3. Specific nucleotide sequences appear necessary to account for the hysteresis observed on titrating ribosomal RNA with acid or alkali within the range pH3.0-7.0 since this phenomenon was less pronounced for Escherichia coli transfer RNA and for RNA from turnip yellow-mosaic virus.
| S-EPMC1264927 | biostudies-other
Project description:The suggested involvement of ribonuclease II in the maturation of rRNA has been examined directly by determining the activity of the enzyme and the amount of p16S rRNA in cell-free extracts from Escherichia coli A19 and its temperature-sensitive derivative N464 grown under experimental conditions designed to vary the amounts of enzyme and precursor independently. In strain A19 the enzyme showed maximum activity in circumstances where the amount of p16S rRNA was normal (e.g. exponential-phase cells) or raised eight times (e.g. during inhibition of growth by methionine starvation of the relaxed auxotroph or by chloramphenicol or puromycin treatment). In strain N464 at the non-permissive temperature the ribonuclease II activity may be decreased by 50% without effect upon the amount of p16S rRNA, whereas in methionine starvation of this strain the enzyme activity is at a maximum and the p16S rRNA is eight times that in exponential-phase cells. These observations are discussed in relation to the previously implied role of ribonuclease II in the maturation of rRNA within ribosome precursors.
| S-EPMC1168021 | biostudies-other
Project description:The RNA of the blue-green alga Anacystis nidulans contains three ribosomal RNA species with molecular weights of 0.56x10(6), 0.9x10(6), and 1.1x10(6) if the RNA is extracted in the absence of Mg(2+). The 0.9x10(6)mol.wt. rRNA is extremely slowly labelled in (32)P-incorporation experiments. This rRNA may be a cleavage product of the 1.1x10(6)mol.wt. rRNA from the ribosomes of cells in certain physiological states (e.g. light-deficiency during growth). The cleavage of the 1.1x10(6)mol.wt. rRNA during the extraction procedure can be prevented by the addition of 10mm-MgCl(2). (32)P-pulse-labelling studies demonstrate the rapid synthesis of two ribosomal precursor RNA species. One precursor RNA migrating slightly slower than the 1.1x10(6)mol.wt. rRNA appears much less stable than the other precursor RNA, which shows the electrophoretic behaviour of the 0.7x10(6)mol.wt. rRNA. Our observations support the close relationship between bacteria and blue-green algae also with respect to rRNA maturation. The conversion of the ribosomal precursor RNA species into 0.56x10(6)- and 1.1x10(6)-mol.wt. rRNA species requires Mg(2+) in the incubation medium.
| S-EPMC1174049 | biostudies-other
Project description:1. The purification of methionyl-transfer-RNA synthetase from Escherichia coli by a modified technique gives a 16% yield of a protein that appears homogeneous by the criteria of disc gel electrophoresis, ultracentrifugation and end-group analysis. 2. The molecular weight is 96000 and the protein consists of two sub-units of 48000, which appear to be identical. The amino acid composition and thiol content are reported. 3. Kinetic data are reported for analogues of methionine and for pure t-RNA(F) and t-RNA(M), which are respectively the methionine transfer RNA that can exist in the formylmethionyl form and the one that can exist only in the methionyl form. The enzyme binds and acylates both species of transfer RNA identically.
| S-EPMC1198805 | biostudies-other
Project description:1. Rapidly labelled RNA from Escherichia coli K 12 was characterized by hybridization to denatured E. coli DNA on cellulose nitrate membrane filters. The experiments were designed to show that, if sufficient denatured DNA is offered in a single challenge, practically all the rapidly labelled RNA will hybridize. With the technique employed, 75-80% hybridization efficiency could be obtained as a maximum. Even if an excess of DNA sites were offered, this value could not be improved upon in any single challenge of rapidly labelled RNA with denatured E. coli DNA. 2. It was confirmed that the hybridization technique can separate the rapidly labelled RNA into two fractions. One of these (30% of the total) was efficiently hybridized with the low DNA/RNA ratio (10:1, w/w) used in tests. The other fraction (70% of the total) was hybridized to DNA at low efficiencies with the DNA/RNA ratio 10:1, and was hybridized progressively more effectively as the amount of denatured DNA was increased. A practical maximum of 80% hybridization of all the rapidly labelled RNA was first achieved at a DNA/RNA ratio 210:1 (+/-10:1). This fraction was fully representative of the rapidly labelled RNA with regard to kind and relative amount of materials hybridized. 3. In competition experiments, where additions were made of unlabelled RNA prepared from E. coli DNA, DNA-dependent RNA polymerase (EC 2.7.7.6) and nucleoside 5'-triphosphates, the rapidly labelled RNA fraction hybridized at a low (10:1) DNA/RNA ratio was shown to be competitive with a product from genes other than those responsible for ribosomal RNA synthesis and thus was presumably messenger RNA. At higher DNA/rapidly labelled RNA ratios (200:1), competition with added unlabelled E. coli ribosomal RNA (without messenger RNA contaminants) lowered the hybridization of the rapidly labelled RNA from its 80% maximum to 23%. This proportion of rapidly labelled RNA was not competitive with E. coli ribosomal RNA even when the latter was in large excess. The ribosomal RNA would also not compete with the 23% rapidly labelled RNA bound to DNA at low DNA/RNA ratios. It was thus demonstrated that the major part of E. coli rapidly labelled RNA (70%) is ribosomal RNA, presumably a precursor to the RNA in mature ribosomes. 4. These studies have shown that, when earlier workers used low DNA/RNA ratios (about 10:1) in the assay of messenger RNA in bacterial rapidly labelled RNA, a reasonable estimate of this fraction was achieved. Criticisms that individual messenger RNA species may be synthesized from single DNA sites in E. coli at rates that lead to low efficiencies of messenger RNA binding at low DNA/RNA ratios are refuted. In accordance with earlier results, estimations of the messenger RNA content of E. coli in both rapidly labelled and randomly labelled RNA show that this fraction is 1.8-1.9% of the total RNA. This shows that, if any messenger RNA of relatively long life exists in E. coli, it does not contribute a measurable weight to that of rapidly labelled messenger RNA.
| S-EPMC1187205 | biostudies-other
Project description:Helix 69 (H69) is a 19-nt stem-loop region from the large subunit ribosomal RNA. Three pseudouridine (Ψ) modifications clustered in H69 are conserved across phylogeny and known to affect ribosome function. To explore the effects of Ψ on the conformations of Escherichia coli H69 in solution, nuclear magnetic resonance spectroscopy was used to reveal the structural differences between H69 with (ΨΨΨ) and without (UUU) Ψ modifications. Comparison of the two structures shows that H69 ΨΨΨ has the following unique features: (i) the loop region is closed by a Watson-Crick base pair between Ψ1911 and A1919, which is potentially reinforced by interactions involving Ψ1911N1H and (ii) Ψ modifications at loop residues 1915 and 1917 promote base stacking from Ψ1915 to A1918. In contrast, the H69 UUU loop region, which lacks Ψ modifications, is less organized. Structure modulation by Ψ leads to alteration in conformational behavior of the 5' half of the H69 loop region, observed as broadening of C1914 non-exchangeable base proton resonances in the H69 ΨΨΨ nuclear magnetic resonance spectra, and plays an important biological role in establishing the ribosomal intersubunit bridge B2a and mediating translational fidelity.
| S-EPMC3973299 | biostudies-literature
Project description:A method is described for measuring the proportion of galactose-specific mRNA (gal-mRNA) in the total RNA extracted from pulse-labelled cells of Escherichia coli K12, by DNA-RNA hybridization with DNA prepared from bacteriophage lambdadg. RNA from wild-type E. coli was compared with RNA from a homogenote carrying the gal operon both in the chromosome and in a substituted sex-factor, and with RNA from a deletion strain that carried the galactose operon only in the exogenote. In each case the cultures were induced with fucose. Under these conditions the amount of gal-mRNA was found to be proportional to the content of galactokinase in the different cultures, and to the gene frequency. The amounts of gal-mRNA in an O(c) mutant and an R(-) mutant were also proportional to the observed contents of galactokinase. In cultures repressed for the enzymes of the galactose operon with thiomethylgalactoside, the content of gal-mRNA was higher than expected from the content of galactokinase. Possible explanations of this finding are discussed.
| S-EPMC1176492 | biostudies-other
Project description:From analyses of the hybridization of Escherichia coli rRNA (ribosomal RNA) to homologous denatured DNA, the following conclusions were drawn. (1) When a fixed amount of DNA was hybridized with increasing amounts of RNA, only 0.35+/-0.02% of E. coli DNA was capable of binding (16s+23s) rRNA. Although preparations of 16s and 23s rRNA were virtually free from cross-contamination, the hybridization curves for purified 16s or 23s rRNA were almost identical with that of the parent specimen containing 1 weight unit of 16s rRNA mixed with 2 weight units of 23s rRNA. The 16s and 23s rRNA also competed effectively for the same specific DNA sites. It appears that these RNA species each possess all hybridizing species typical of the parent (16s+23s) rRNA specimen, though probably in different relative amounts. (2) By using hybridization-efficiency analysis of DNA-RNA hybridization curves (Avery & Midgley, 1969) it was found that (a) 0.45% of the DNA would hybridize total rRNA and (b) when so little RNA was added to unit weight of DNA that the DNA sites were not saturated, only 70-75% of the input RNA would form hybrids. The reasons for the discrepancy between the results obtained by the two alternative analytical approaches were discussed. (3) For either 16s or 23s rRNA, hybridization analysis indicated that two principal weight fractions of rRNA may exist, hybridizing to two distinct groups of DNA sites. However, these groups seem to be incompletely divided between the 16s and 23s fractions. Analysis suggested that (a) 85% of the 16s rRNA was hybridized to about half the DNA that specifically binds rRNA (0.23% of the total DNA). (b) 70% of the 23s rRNA hybridized to a further 0.23% of the DNA and (c) the minor fraction (15%) of 16s rRNA may be competitive with the major fraction (70%) of 23s rRNA. Conversely, the minor fraction (30%) of the 23s rRNA may compete with the major fraction (85%) of 16s rRNA. Models were proposed to explain the apparent lack of segregation of distinct RNA species in the two subfractions of rRNA. (4) If protein synthesis and ribosome maturation were inhibited in cells of an RC(rel) mutant, E. coli W 1665, by depriving them of an amino acid (methionine) essential for growth, the inhibition had no discernible effect on the relative rates of synthesis of rRNA species. The rRNA that accumulates in RC(rel) strains of E. coli after amino acid deprivation is apparently identical in its content of RNA species with that of the pre-existing mature RNA in the ribosomes. On the other hand, the messenger RNA is stabilized, and accumulates as about 15% of the RNA formed after withdrawal of the amino acid.
| S-EPMC1185118 | biostudies-other
Project description:The concept of promoter efficiency is introduced as frequency of RNA chain initiation at a given promoter normalized to the intracellular concentration of free (but functional) RNA polymerase. Previous observations from this laboratory on the synthesis of ribosomes and beta-galactosidase are used to show that during a nutritional shift-up from succinate minimal to glucose-amino acids medium (3-fold increase in steady-state growth rate) the concentration of free (active) RNA polymerase decreases to one-quarter of the pre-shift value and the promoter efficiencies of the genes for ribosomal RNA and ribosomal proteins increase 9- and 6-fold respectively. This extent of control of ribosomal genes is much greater than expected on the basis of the increase in the rate of ribosome synthesis (3-fold).
| S-EPMC1165697 | biostudies-other