The messenger ribonucleic acid content of Bacillus subtilis 168

Bacillus subtilis 168 messenger RNA was determined by DNA–RNA hybridization techniques, with denatured DNA immobilized upon cellulose nitrate membrane filters. The following results were obtained. (1) Cultures of B. subtilis, growing exponentially in enriched glucose–salts medium at 37°, incorporated [5−3H]uracil into both ribosomal and messenger RNA fractions without the kinetic delay expected from the presence of the intracellular nucleotide pools. (2) However short the time of labelling with exogenous labelled uracil (down to 7sec.), 32–36% of the rapidly labelled RNA was messenger RNA and 68–64% was an RNA with the hybridization characteristics of ribosomal RNA. Analysis of the apparent nucleotide base composition of total 32P-labelled rapidly labelled RNA and the two RNA fractions separated by hybridization at a DNA/RNA ratio 5:1 confirmed this finding. Of the rapidly labelled RNA, 31% readily hybridized with DNA at low DNA/RNA ratios and had an apparent base composition like that of the DNA, whereas 69% was hybridized only at low efficiency at low DNA/RNA ratios and had a composition identical with that of ribosomal RNA. (3) In cultures dividing every 48min. at 37°, kinetic analysis of RNA labelled over a 20min. period showed that the average life-time of messenger RNA was 2·7–3·0min. and that its amount was 3·0% of the total RNA. (4) The hybridization of 3H-labelled randomly labelled RNA with DNA at a DNA/RNA ratio 5:1 showed that 2·9% of the randomly labelled RNA had the characteristics of messenger RNA. (5) Experiments carried out as described by Pigott & Midgley (1968) indicated that hybridization at low DNA/RNA ratios (5:1) effectively accounted for all the messenger RNA in a given specimen. The efficiency coefficient of RNA hybridization lay within the range of 90–95% input, if an excess of DNA sites was offered for RNA binding. (6) These measurements are compared with other results obtained by different methods, and reasons for any major disagreement are suggested.

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Characterization of rapidly labelled ribonucleic acid in Escherichia coli by deoxyribonucleic acid–ribonucleic acid hybridization

Biochemical Journal ◽

10.1042/bj1100251 ◽

1968 ◽

Vol 110 (2) ◽

pp. 251-263 ◽

Cited By ~ 27

Author(s):

G. H. Pigott ◽

J. E. M. Midgley

Keyword(s):

Escherichia Coli ◽

Ribosomal Rna ◽

Ribonucleic Acid ◽

Messenger Rna ◽

Rna Binding ◽

Hybridization Technique ◽

E Coli ◽

K 12 ◽

Dna 2

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.

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Synthesis and turnover of mitochondrial ribonucleic acid in HeLa cells: the mature ribosomal and messenger ribonucleic acid species are metabolically unstable.

Molecular and Cellular Biology ◽

10.1128/mcb.1.6.497 ◽

1981 ◽

Vol 1 (6) ◽

pp. 497-511 ◽

Cited By ~ 132

Author(s):

R Gelfand ◽

G Attardi

Keyword(s):

Ribosomal Rna ◽

Hela Cells ◽

Ribonucleic Acid ◽

Half Life ◽

Messenger Rna ◽

Specific Activity ◽

Mitochondrial Rna ◽

Messenger Rnas ◽

Precursor Pool ◽

Messenger Ribonucleic Acid

The synthesis rates and half-lives of the individual mitochondrial ribosomal ribonucleic acid (RNA) and polyadenylic acid-containing RNA species in HeLa cells have been determined by analyzing their kinetics of labeling with [5-3H]-uridine and the changes in specific activity of the mitochondrial nucleotide precursor pools. In one experiment, a novel method for determining the nucleotide precursor pool specific activities, using nascent RNA chains, has been utilized. All mitochondrial RNA species analyzed were found to be metabolically unstable, with half-lives of 2.5 to 3.5 h for the two ribosomal RNA components and between 25 and 90 min for the various putative messenger RNAs. A cordycepin "chase" experiment yielded half-life values for the messenger RNA species which were, in general, larger by a factor of 1.5 to 2.5 than those estimated in the labeling kinetics experiments. On the basis of previous observations, a model is proposed whereby the rate of mitochondrial RNA decay is under feedback control by some mechanism linked to RNA synthesis or processing. A short half-life was determined for five large polyadenylated RNAs, which are probably precursors of mature species. A rate of synthesis of one to two molecules per minute per cell was estimated for the various H-strand-coded messenger RNA species, and a rate of synthesis 50 to 100 times higher was estimated for the ribosomal RNA species. These data indicate that the major portion of the H-strand in each mitochondrial deoxyribonucleic acid molecule is transcribed very infrequently, possibly as rarely as once or twice per cell generation. Furthermore, these results are consistent with a previously proposed model of H-strand transcription in the form of a single polycistronic molecule.

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Accumulation of Messenger Ribonucleic Acid Specific for Extracellular Protease in Bacillus subtilis 168

Journal of Bacteriology ◽

10.1128/jb.116.2.531-534.1973 ◽

1973 ◽

Vol 116 (2) ◽

pp. 531-534 ◽

Cited By ~ 11

Author(s):

E. V. Semets ◽

A. R. Glenn ◽

B. K. May ◽

W. H. Elliott

Keyword(s):

Bacillus Subtilis ◽

Ribonucleic Acid ◽

Extracellular Protease ◽

Messenger Ribonucleic Acid ◽

Bacillus Subtilis 168

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Lethal effect of rifampicin in Bacillus subtilis as a complicating factor in the assessment of the lifetime of messenger ribonucleic acid

Biochemical Journal ◽

10.1042/bj1340263 ◽

1973 ◽

Vol 134 (1) ◽

pp. 263-270 ◽

Cited By ~ 13

Author(s):

John G. Coote ◽

David A. Wood ◽

Joel Mandelstam

Keyword(s):

Bacillus Subtilis ◽

Phase Contrast ◽

Actinomycin D ◽

Lethal Effect ◽

Cell Integrity ◽

Bactericidal Action ◽

Physical Damage ◽

Messenger Ribonucleic Acid ◽

Bacillus Subtilis 168 ◽

Synchronized Cultures

The bactericidal action of rifampicin was compared with that of chloramphenicol in growing and in sporulating cultures of Bacillus subtilis 168. Chloramphenicol kills cells only very slowly, but exposure to rifampicin kills over 95% of cells in a few minutes, causing gross physical damage, which is visible in both phase-contrast and electron microscopy. This is accompanied by a fall in O2 consumption and by lysis. Experiments with synchronized cultures showed that susceptibility to the lethal effect of rifampicin is greater when the cells are dividing. The results suggest that the synthesis of some species of RNA other than mRNA may be necessary for the maintenance of cell integrity, although experiments with actinomycin D do not altogether fit this interpretation. However, we conclude that rifampicin is too toxic to use as an antibiotic for assessing the lifetime of mRNA.

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The control of ribonucleic acid synthesis in bacteria. Steady-state content of messenger ribonucleic acid in Escherichia coli M.R.E. 600

Biochemical Journal ◽

10.1042/bj1200279 ◽

1970 ◽

Vol 120 (2) ◽

pp. 279-288 ◽

Cited By ~ 9

Author(s):

W. J. H. Gray ◽

J. E. M. Midgley

Keyword(s):

Escherichia Coli ◽

Ribosomal Rna ◽

Ribonucleic Acid ◽

Messenger Rna ◽

Acid Synthesis ◽

Growth Conditions ◽

Transfer Rna ◽

Average Lifetime ◽

Messenger Ribonucleic Acid ◽

Wide Range

1. The technique of DNA–RNA hybridization was used to follow changes in the amount and average lifetime of unstable messenger RNA in Escherichia coli M.R.E. 600 over a wide range of different growth conditions. The method of analysis was based on the kinetics of incorporation of exogenous labelled nucleic acid bases into the RNA of steadily growing cultures, as described by Bolton & McCarthy (1962). 2. The ratio of the average lifetime of messenger RNA to the mean generation time of E. coli cultures was constant over the temperature range 25–45°C in a given medium, but the constant varied with the nature of the growth medium. For cultures growing in sodium lactate–salts or glucose–salts media the ratio was 0.046±0.005 and in enriched broth it was 0.087±0.009. Measurements of the amounts of transfer RNA, ribosomal RNA and messenger RNA were also made. The results confirmed earlier reports that the ratio of the amount of messenger RNA to the amount of ribosomes in the cells is virtually constant. On the other hand, the ratio of the amount of transfer RNA to the amount of ribosomal RNA decreased with increasing growth rate at a given temperature. 3. In cultures at temperatures higher than necessary for optimum rates of growth the average lifetime of messenger RNA lengthened in harmony with the increased time required for cell division. It seems that suboptimum growth rates at higher temperatures cannot be explained simply as a combination of increased rates of synthesis and breakdown of messenger RNA with a grossly decreased efficiency of translation. The absolute rate of messenger RNA synthesis was lowered, and its amount in the cells was typical of all other cultures grown at lower temperatures in the same medium. 4. The rate of entry of exogenous labelled uracil into unstable messenger RNA and stable ribosomal RNA was constant in all media at all temperatures in the approximate ratio 1:2. In media supporting a lower rate of growth, e.g. lactate–salts or glucose–salts media, the messenger RNA fraction constituted 2.2±0.3% of the total cellular RNA. In enriched broth 3.6±0.3% of the total RNA was messenger.

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Synthesis and turnover of mitochondrial ribonucleic acid in HeLa cells: the mature ribosomal and messenger ribonucleic acid species are metabolically unstable

Molecular and Cellular Biology ◽

10.1128/mcb.1.6.497-511.1981 ◽

1981 ◽

Vol 1 (6) ◽

pp. 497-511

Author(s):

R Gelfand ◽

G Attardi

Keyword(s):

Ribosomal Rna ◽

Hela Cells ◽

Ribonucleic Acid ◽

Half Life ◽

Messenger Rna ◽

Specific Activity ◽

Mitochondrial Rna ◽

Messenger Rnas ◽

Precursor Pool ◽

Messenger Ribonucleic Acid

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A method for measuring the content of a specific messenger ribonucleic acid in Escherichia coli

Biochemical Journal ◽

10.1042/bj1210105 ◽

1971 ◽

Vol 121 (1) ◽

pp. 105-108 ◽

Cited By ~ 3

Author(s):

J. O. Bishop ◽

M. I. Irving

Keyword(s):

Escherichia Coli ◽

Ribosomal Rna ◽

Ribonucleic Acid ◽

Messenger Rna ◽

Model System ◽

Total Rna ◽

Messenger Ribonucleic Acid

A method is described for measuring the porportion of a specific messenger RNA in the total RNA extracted from pulse-labelled cells. A model system consisting of total ribosomal RNA and Escherichia coli DNA is used to validate the method and to define the conditions under which it can be used.

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Messenger ribonucleic acid content of Bacillus amyloliquefaciens throughout its growth cycle compared with Bacillus subtilis 168

Journal of Molecular Biology ◽

10.1016/0022-2836(75)90353-8 ◽

1975 ◽

Vol 96 (2) ◽

pp. 345-352 ◽

Cited By ~ 13

Author(s):

S. Brown ◽

G. Coleman

Keyword(s):

Bacillus Subtilis ◽

Ribonucleic Acid ◽

Acid Content ◽

Bacillus Amyloliquefaciens ◽

Growth Cycle ◽

Messenger Ribonucleic Acid ◽

Bacillus Subtilis 168

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Messenger RNA Turnover Processes inEscherichia coli, Bacillus subtilis, and Emerging Studies inStaphylococcus aureus

International Journal of Microbiology ◽

10.1155/2009/525491 ◽

2009 ◽

Vol 2009 ◽

pp. 1-15 ◽

Cited By ~ 47

Author(s):

Kelsi L. Anderson ◽

Paul M. Dunman

Keyword(s):

Gene Expression ◽

Bacillus Subtilis ◽

Mrna Stability ◽

Messenger Rna ◽

Binding Proteins ◽

Target Genes ◽

Rna Binding ◽

Rna Binding Proteins ◽

Mrna Turnover ◽

Small Noncoding Rnas

The regulation of mRNA turnover is a recently appreciated phenomenon by which bacteria modulate gene expression. This review outlines the mechanisms by which three major classes of bacterialtrans-acting factors, ribonucleases (RNases), RNA binding proteins, and small noncoding RNAs (sRNA), regulate the transcript stability and protein production of target genes. Because the mechanisms of RNA decay and maturation are best characterized inEscherichia coli, the majority of this review will focus on how these factors modulate mRNA stability in this organism. However, we also address the effects of RNases, RNA binding proteins, sRNAs on mRNA turnover, and gene expression inBacillus subtilis, which has served as a model for studying RNA processing in gram-positive organisms. We conclude by discussing emerging studies on the role modulating mRNA stability has on gene expression in the important human pathogenStaphylococcus aureus.

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