The control of ribonucleic acid synthesis in bacteria. The synthesis and stability of ribonucleic acid in chloramphenicol-inhibited cultures of Escherichia coli

The rate of polymerization of ribosomal ribonucleic acid chains was estimated for steadily growing cultures of Escherichia coli M.R.E.600, from the kinetics of incorporation of exogenous [5-³H]uracil into completed 23S rRNA molecules. The analytical method of Avery & Midgley (1971) was used. Measurements were made at 37°C, in the presence or the absence of chloramphenicol, in each of three media; enriched broth, glucose–salts or sodium lactate–salts. The rate of chain elongation of 23S rRNA was virtually constant in all media at 37°C, as 24±4 nucleotides added/s. Accelerations in the rate of biosynthesis of rRNA by chloramphenicol in growth-limiting media are due primarily to an increase in the rate of initiation of new RNA chains, up to the rates existing in cultures growing rapidly in broth. Thus, in poorer media, only a small fraction of the available DNA-dependent RNA polymerase molecules are active at any given instant, since the chain-initiation rate is limiting in these conditions. In cultures growing rapidly in enriched broth, antibiotic inhibition caused a rise of some 12% in the rate of incorporation of exogenous uracil into total RNA. This small acceleration was due entirely to the partial stabilization of the mRNA fraction, which accumulated as 14% of the RNA formed after the addition of chloramphenicol. In cultures growing more slowly in glucose–salts or lactate–salts media, chloramphenicol caused an immediate acceleration of two- to three-fold in the overall rate of RNA synthesis. Studies by DNA–RNA hybridization showed that the synthesis of mRNA was accelerated in harmony with the other affected species. However, just over half the mRNA formed after the addition of chloramphenicol quickly decayed to acid-soluble products, whereas the remainder was more stable and accumulated in the cells. The mRNA fraction constituted about 6% of the total cellular RNA after 3h inhibition. A model was suggested to explain the partial stabilization and accumulation of the mRNA fraction and the acceleration in the rate of synthesis of mRNA when chloramphenicol was added to cultures in growth-limiting media.     

Fingerprinting nonradioactive ribonucleic acid with the aid of polynucleotide phosphorylase

We describe a method for obtaining radioactive fingerprints from nonradioactive ribonucleic acid. Fragments derived by T1 ribonuclease digestion of RNA are dephosphorylated with bacterial alkaline phosphatase. When these fragments are used as primers for the reaction of primer dependent polynucleotide phosphorylase with [α-³²P]GDP in the presence of T1 ribonuclease the 3′-hydroxyl group of each fragment becomes phosphorylated. The degree of phosphorylation is reasonably uniform. The method has been applied to T1 ribonuclease digests of Escherichia coli tRNA^Met_f; the oligonucleotides were further analyzed by spleen phosphodiesterase digestion. In a similar manner fingerprints of pancreatic ribonuclease digests of RNA can be obtained, when [α-³²P]UDP, polynucleotide phosphorylase and pancreatic ribonuclease are used.     

The control of ribonucleic acid synthesis in bacteria. The synthesis and stability of ribonucleic acids in relaxed and stringent amino acid auxotrophs of Escherichia coli

The biosynthesis and stability of various RNA fractions was studied in RC(str) and RC(rel) multiple amino acid auxotrophs of Escherichia coli. In conditions of amino acid deprivation, RC(str) mutants were labelled with exogenous nucleotide bases at less than 1% of the rate found in cultures growing normally in supplemented media. Studies by DNA-RNA hybridization and by other methods showed that, during a period of amino acid withdrawal, not more than 60-70% of the labelled RNA formed in RC(str) mutants had the characteristics of mRNA. Evidence was obtained for some degradation of newly formed 16S and 23S rRNA species to heterogeneous material of lower molecular weight. This led to overestimations of the mRNA content of rapidly labelled RNA from such methods as simple examination of sucrose-density-gradient profiles. In RC(rel) strains the absolute and relative rates of synthesis of the various RNA fractions were not greatly affected. However, the stability of about half of the mRNA fraction was increased in RC(rel) strains during amino acid starvation, giving kinetics of mRNA labelling and turnover that were identical with those found in either RC(str) or RC(rel) strains inhibited by high concentrations of chloramphenicol. Coincidence hybridization techniques showed that the mRNA content of amino acid-starved RC(str) auxotrophs was unchanged from that found in normally growing cells. In contrast, RC(rel) strains deprived of amino acids increased their mRNA content about threefold. In such cultures the mRNA content of accumulating newly formed RNA was a constant 16% by wt.     

A method for determination of the synthesis rate of stable and unstable ribonucleic acid in Escherichia coli

A method to determine the relative rates of stable and unstable RNA synthesis is presented. The method employs the antibiotic rifampicin. For exponential phase Escherichia coli B/r growing at rates between 0.67 and 2.10 doublings per hr (3.2-fold range in growth rate), the fraction of the instantaneous rate of total RNA synthesis which is stable RNA was found to increase 2.3-fold (with increasing growth rate) from 0.29 to 0.68. Correspondingly, the fraction of the instantaneous rate of RNA synthesis which is unstable and presumably mRNA, decreases from 0.71 to 0.32 over this growth rate range.These relative synthesis rates are in good agreement with values obtained by DNA-RNA hybridization and other methods reported in the literature. The new method is simpler than these other methods and presumably as accurate.     

The effect of trimethoprim on macromolecular synthesis in Escherichia coli. General effects on ribonucleic acid and protein synthesis

In trimethoprim-inhibited RC(str) strains of Escherichia coli, the expression of the RC control of stable RNA synthesis arose primarily from a decrease in the intracellular concentrations of glycine and methionine, and not from inhibition of the initiation of new protein chains. In non-supplemented cultures, experiments with rifampicin showed that the immediate response to the addition of trimethoprim was a rapid decrease in the rate of initiation of RNA chains. This was followed after a few minutes by a sufficiently large fall in the rate of endogenous synthesis of nucleotide bases to cause a decrease in the rate of RNA chain polymerization. Inhibition of RNA chain initiation was thus overridden by an accumulation of DNA-dependent RNA polymerases upon the cistrons. RC(rel) strains also accumulated polymerases upon the DNA in similar circumstances, but did not suffer the initial effects on chain initiation. If purines were supplied before adding trimethoprim, RC(str) strains polymerized RNA chains at normal rates, but initiation rates were permanently decreased. In either situation, an increased% of the RNA formed was mRNA. However, in RC(rel) strains supplemented with bases, trimethoprim did not affect either the rate of initiation of new chains or their rates of polymerization or the relative rates of synthesis of stable RNA and mRNA. Protein synthesis was also severely inhibited by trimethoprim. Though the addition of glycine and methionine to base-supplemented, trimethoprim-inhibited RC(str) strains did not apparently affect the decreased rate of protein synthesis, RNA accumulation resumed at its normal rate. Thus, the inhibition of protein chain initiation had no effect on the rate of RNA accumulation in either RC(str) or RC(rel) bacteria. The RC control does not express itself through inhibitions of protein synthesis at this level.     

Ribosomal ribonucleic acid synthesis in Bacillus subtilis

The mode of biosynthesis of the 16S and 23S ribosomal ribonucleic acids (rRNA) was studied in Bacillus subtilis 168thy(-). Three criteria were used to define the characteristics of the rRNA species: (i) the time required at 37 degrees C to synthesize 16S and 23S rRNA chains de novo in growing cultures; (ii) the degree of reactivity of the 3'-terminal groups of the rRNA molecules with periodate and [carbonyl-(14)C]isonicotinic acid hydrazide; and (iii) the reactivity of the 5'-terminal regions of the rRNA molecules with the bacterial exonuclease purified by Riley (1969). The 16S and 23S chains of B. subtilis were synthesized at rates of 22+/-2 and 21+/-2 nucleotides added/s. The periodate-[(14)C]isonicotinic acid hydrazide and the exonuclease techniques for estimating apparent chain lengths of RNA indicated that the chain length of the 23S rRNA was 1.8 times that of the 16S fraction. The apparent chain lengths of each rRNA species were: 16S rRNA, 1650+/-50 nucleotide residues; 23S rRNA, 3050+/-90 nucleotide residues. It appears that, the 16S and 23S rRNA molecules in B. subtilis are synthesized in the expected manner, by simple polymerization of the final products on independent cistrons.     

Rifampicin Inhibition of Ribonucleic Acid and Protein Synthesis in Normal and Ethylenediaminetetraacetic Acid-Treated Escherichia coli

The kinetics of ribonucleic acid (RNA) and protein synthesis in rifampicin-inhibited normal and ethylenediaminetetraacetic acid (EDTA)-treated Escherichia coli was measured. Approximately 200-fold higher external concentrations of rifampicin were needed to produce a level of inhibition in normal cells comparable to that observed in EDTA-treated cells. The rates of RNA and protein synthesis in both kinds of cells decreased exponentially, after an initial lag phase, at all rifampicin concentrations tested. The lag phase was longer and the final exponential slope less for protein synthesis than for RNA synthesis at a given rifampicin concentration. Below certain rifampicin concentrations, both the lag phase and the subsequent exponential decrease in the rates of RNA and protein synthesis were found to be rifampicin concentration dependent. At greater concentrations only the time of the lag phase was decreased by higher rifampicin concentrations, whereas the slope of the exponential decrease in the rates of RNA and protein synthesis was unaffected. In all cases, the exponential decrease continued to at least a 99.8% inhibition of the original rate of synthesis. These in vivo results are consistent with the mode of rifampicin action determined from in vitro studies; rifampicin prevents initiations of RNA polymerase on deoxyribonucleic acid, but not its propagation, by binding the enzyme essentially irreversibly. The results also indicate the size distribution of messenger RNA molecules in E. coli under our conditions.     

Incorporation of cytokinin N-benzyladenine into tobacco callus transfer ribonucleic Acid and ribosomal ribonucleic Acid preparations

The incorporation of the cytokinin N⁶-benzyladenine into tobacco (Nicotiana tabacum) callus tRNA and rRNA preparations isolated from tissue grown on medium containing either N⁶-benzyladenine-8-¹⁴C or N⁶-benzyladenine-8-¹⁴C: benzene-³H(G) has been examined. N⁶-benzyladenine was incorporated into both the tRNA and rRNA preparations as the intact base. Over 90% of the radioactive N⁶-benzyladenosine recovered from the RNA preparations was associated with the rRNA. Purification of the crude rRNA by either MAK chromatography or Sephadex G-200 gel filtration had no effect on the N⁶-benzyladenosine content of the RNA preparation. The distribution of N⁶-benzyladenosine moieties in tobacco callus tRNA fractionated by BD-cellulose chromatography did not correspond to the distribution of ribosylzeatin activity. N⁶-benzyladenosine was released from the rRNA preparation by treatment with venom phosphodiesterase and phosphatase, ribonuclease T₂ and phosphatase, or ribonuclease T₂ and a 3′-nucleotidase. N⁶-benzyladenosine was not released from the RNA preparation by treatment with either ribonuclease T₂ or phosphatase alone or by successive treatment with ribonuclease T₂ and a 5′-nucleotidase. Brief treatment of the rRNA preparation with ribonuclease T₁ and pancreatic ribonuclease converted the N⁶-benzyladenosine moieties into an ethyl alcohol soluble form. On the basis of these and earlier results, the N⁶-benzyladenosine recovered from the tobacco callus RNA preparations appears to be present as a constituent of RNA and not as a nonpolynucleotide contaminant.     

Phage formation in Staphylococcus muscae cultures. XI. The synthesis of ribonucleic acid,desoxyribonucleic acid,and protein in uninfected bacteria

1. The synthesis of ribonucleic acid, desoxyribomicleic acid, and protein in S. muscae has been studied: (a) during the lag phase, (b) during the early log phase, and (c) while the cells are forming an adaptive enzyme for lactose utilization. 2. During the lag phase there may be a 60 per cent increase in ribonucleic acid and protein and a 50 per cent increase in dry weight without a change in cell count, as determined microscopically, or an increase in turbidity. 3. During this period, the increase in protein closely parallels the increase in ribonucleic acid, in contrast to desoxyribonucleic acid, which begins to be synthesized about 45 minutes after the protein and ribonucleic acid have begun to increase. 4. The RNA N/protein N ratio is proportional to the growth rate of all S. muscae strains studied. 5. While the RNA content per cell during the early log phase depends upon the growth rate, the DNA content per cell is fairly constant irrespective of the growth rate of the cell. 6. Resting cells of S. muscae have approximately the same RNA content per cell irrespective of their prospective growth rate. 7. While the cells are adapting to lactose, during which time there is little or no cellular division, there is never an increase of protein without a simultaneous increase in ribonucleic acid, the RNA N/protein N ratio during these intervals being approximately 0.15. 8. Lactose-adapting cells show a loss of ribonucleic acid. The purines-pyrimidines of the ribonucleic acid can be recovered in the cold 5 per cent trichloroacetic acid fraction, but the ribose component is completely lost from the system. 9. The significance of these results is discussed in relation to the importance of ribonucleic acid for protein synthesis.     

Stability of rapidly labelled messenger ribonucleic acid in Bacillus amyloliquefaciens during the phases of minimum and maximum extracellular enzyme formation.

The stability of rapidly labelled hybridizable messenger RNA in both exponential and post-exponential phase cells of Bacillus amyloliquefaciens was measured in terms of the rate of loss of its radioactivity. In the exponential phase, where 96% of the mRNA was specific for cell proteins and only 4% was exoprotein mRNA, the label was lost exponentially from the rapidly labelled hybridizable mRNA fraction with a half-life of six minutes at 30 °C. The antibiotic rifampicin, at a concentration of 10 μg/ml, had no effect on the characteristics of decay of this exponential-phase mRNA. In the post-exponential phase, where there were equal amounts of cell protein and exoprotein-specific mRNA, rapidly labelled hybridizable mRNA decayed exponentially in the presence of rifampicin (10 μg/ml), with a half-life of six minutes at 30 °C. In the absence of rifampicin the characteristics of decay were more complex. The evidence available suggested that this was due to the superimposition of a component attributable to reincorporation of degradation products of radioactive RNA on the characteristic exponential decay pattern of the post-exponential mRNA.Measurement of the stability of active mRNA, by studying the loss of ability to incorporate l-[¹⁴C]leucine into protein in the presence of rifampicin (10 μg/ml), gave half-lives of 4.5 minutes and six minutes, respectively, for exponential and post-exponential material.     

The control of ribonucleic acid synthesis in bacteria. Fluctuations in messenger ribonucleic acid synthesis in cultures recovering from amino acid starvation

Changes in the cell content and rate of synthesis of mRNA were studied in auxotrophs of Escherichia coli recovering from a period of amino acid deprivation. Parallel studies were carried out on bacterial strains inhibited with trimethoprim, when glycine and methionine were added to relieve an amino acid deficiency. In the latter case, protein synthesis was still severely inhibited through a lack of N-formylmethionyl-tRNA(fMet) for chain initiation, so that fewer ribosomes were attached to mRNA chains. (1) In RC(str) strains recovering from amino acid starvation, there was a transient oversynthesis of mRNA, but the amounts returned to normal after about a 15-min period of recovery. RC(rel) strains did not show this effect; any extra mRNA accumulated during the previous starvation period was rapidly lost, but no oversynthesis occurred during the resumption of growth. (2) In trimethoprim-inhibited cultures supplemented with glycine and methionine, mRNA was produced at the same rate, relative to stable RNA species, as during normal growth. The evidence implied that decreased rates of ribosome attachment had no effect on the functional or chemical lifetime of the mRNA fraction. This suggests that mRNA stability does not depend on the frequency of translation by ribosomes. (3) Changes in the mRNA contents of trimethoprim-inhibited RC(str) and RC(rel) cultures were noted soon after supplementation with glycine and methionine. These closely followed those observed in cultures recovering from simple amino acid withdrawal.     

Covalent linkage between ribonucleic Acid primer and deoxyribonucleic Acid product of the avian myeloblastosis virus deoxyribonucleic Acid polymerase

Initiation of deoxyribonucleic acid (DNA) synthesis by the avian myeloblastosis virus DNA polymerase was previously suggested to involve a ribonucleic acid (RNA) primer, the initial product being a DNA molecule joined by a phosphodiester bond to the RNA primer. The existence and nature of such an RNA-DNA joint was investigated by assaying for transfer of a ³²P atom from an α-³²P-deoxyribonucleotide to a 2′(3′)-ribonucleotide after alkaline hydrolysis of the polymerase product. Such a transfer was observed, but only from α-³²P-deoxyadenosine triphosphate and only to 2′(3′)-adenosine monophosphate. This same transfer was observed in both the endogenous DNA polymerase reaction of purified virions and the reconstructed reaction of purified DNA polymerase plus purified 60 to 70S viral RNA. These results indicate a high level of specificity for the initiation process and support the idea of a low-molecular-weight initiator RNA as part of the 60 to 70S RNA complex.     

Protective activities of ribosomal ribonucleic acid and lipopolysaccharide of Pseudomonas aeruginosa: a comparative study

Ribosomal ribonucleic acid (RNA) and lipopolysaccharide (LPS) from P. aeruginosa were compared with respect to their protective activities in mice against an infection with P. aeruginosa. This study is concentrated on the protective activity of RNA. RNA isolated from purified ribosomes did not contain LPS as determined with the Limulus test. Injection of RNA with the adjuvant dimethyldioctadecylammonium bromide (DDA) protected mice against P. aeruginosa without inducing LPS-specific antibodies. C₃H/HeJ mice which are relatively insensitive to the protective activity of LPS could be protected with RNA. The protective activities of RNA and LPS from a mutant strain of P. aeruginosa, PAC 605, containing defective lipopolysaccharide, were compared with the protective activities of RNA and LPS from the parent strain, PAC IR. The protective activity of LPS from PAC 605 was 1000 fold lower than the protective activity of LPS from PAC IR. RNA preparations of both strains induced similar percentages of survival. The protective activity of ribosomal RNA from P. aeruginosa was nonspecific since mice were also protected against a heterologous serotype of P. aeruginosa and against Escherichia coli. RNA from ribosomes of P. aeruginosa, E. coli and the non-lipopolysaccharide containing Saccharomyces cerevisiae had similar protective activities. No protection was obtained with the ribonucleic acid from the E. coli phage MS 2. It is concluded that ribosomal RNA has protective activities distinct from those of LPS.     

RNA polymerase is required for DNA initiation in vitro

Summary We have previously reported in vitro complementation assays for chromosome initiation that enable dnaA and dnaC mutant extracts to synthesize DNA. To examine the role of RNA polymerase in chromosome initiation, inhibitors of the enzyme and anti-RNA polymerase antibody were used. Though rifampicin failed to efficiently inhibit ribonucleoside triphosphate polymerization under the assay conditions, both streptolydigin and anti-RNA plymerase antibody abolished ribonucleic acid synthesis completely. Antibody effectively inhibited chromosome initiation in the dnoA mutant based reaction but streptolydigin did not. Neither streptolydigin nor antibody affected the dnaC-dependent assay. It was concluded that RNA polymerase is required for initiation but not necessarily to polymerize a polyribonucleotide. A scheme for the sequence of initiation events is presented.     

Ribosomal ribonucleic acid maturation in synchronous strain l cells

Summary The incorporation of [³H]-5-uridine into cytoplasmic 18S and 28S ribosomal ribonucleic acid (rRNA) was examined in Colcemid-synchronized strain L cells during G1 and S phases of the cell cycle in the presence of 5×10⁻⁵ m uridine, which was determined to be the saturating concentration for this system. The data show that in S phase a significant increase occurs in the level of [³H]-5-uridine incorporation into each rRNA species. During a 90-min exposure period, S phase cells incorporate 3.4 times as much [³H]-5-uridine into 18S rRNA and 1.9 times as much into 28S rRNA as do G1 cells. The time required for maturation of the ribosomal RNA species during G1 and during S phase is the same, with 18S rRNA appearing in the cytoplasm in 20 min and 28S rRNA in 40 min.     

Characterization of S-Adenosylmethionine: Ribosomal Ribonucleic Acid-Adenine (N6-) Methyltransferase of Escherichia coli Strain B

This study is concerned with the isolation and characterization of the enzyme, S-adenosylmethionine:ribosomal ribonucleic acid-adenine (N⁶⁻) methyl-transferase [rRNA-adenine (N⁶-) methylase] of Escherichia coli strain B, which is responsible for the formation of N⁶-methyladenine moieties in ribosomal ribonucleic acids (rRNA). A 1,500-fold purified preparation of the species-specific methyltransferase methylates a limited number of adenine moieties in heterologous rRNA (Micrococcus lysodeikticus and Bacillus subtilis) and methyl-deficient homologous rRNA. The site recognition mechanism does not require intact 16 or 23S rRNA. The enzyme does not utilize transfer ribonucleic acid as a methyl acceptor nor does it synthesize 2-methyladenine or N⁶-dimethyladenine moieties. Mg²⁺, spermine, K⁺, and Na⁺ increase the reaction rate but not the extent of methylation; elevated concentrations of the cations inhibit markedly. The purified preparations utilize 9-β-ribosyl-2,6-diaminopurine (DAPR) as a methyl acceptor with the synthesis of 9-β-ribosyl-6-amino-2-methylaminopurine. A comparison of the two activities demonstrated that one methyltransferase is responsible for the methylation of both DAPR and rRNA. This property provides a sensitive assay procedure unaffected by ribonucleases and independent of any specificity exhibited by rRNA methyl acceptors.     

Role of aminoacyl-transfer ribonucleic acid in the regulation of ribonucleic acid synthesis in Escherichia coli

Morris, D. W. (University of California, San Diego), and J. A. DeMoss. Role of aminoacyl-transfer ribonucleic acid in the regulation of ribonucleic acid synthesis in Escherichia coli. J. Bacteriol. 90:1624-1631. 1965.-A leucine auxotroph of Escherichia coli was examined for its rate of ribonucleic acid (RNA) synthesis and the level of charged leucine-, arginine-, and valine-specific transfer RNA (tRNA) during the exponential growth period and when growth was limited by leucine starvation. During the logarithmic growth period, the leucine-specific tRNA was 70% charged, arginine-specific tRNA was 30% charged, and the valine-specific tRNA was 80% charged. When leucine became limiting, RNA synthesis was inhibited and the levels of charged arginine- and valine-specific tRNA remained constant, whereas the level of charged leucine-specific tRNA dropped to 40%. Examination of the leucyl-tRNA during the leucine starvation period showed that this 40% level is maintained by protein turnover. Addition of chloramphenicol or puromycin to a leucine-starved culture derepressed RNA synthesis. In the presence of chloramphenicol, the leucine-specific tRNA was fully charged; however, in the presence of puromycin the amount of charged leucine-specific tRNA remained at the starved level. Therefore, during leucine starvation the level of uncharged leucine-specific tRNA is not invariably correlated with the rate of RNA synthesis. We propose that it is the availability of charged tRNA and not the amount of uncharged tRNA which is the important factor in the amino acid control of RNA synthesis.     

Characteristics of pulse-labeled ribonucleic acid in relation to medium pH and acetate concentration during meiosis of yeast

Summary Cellular impermeability associated with sporulating cells of Saccharomyces cerevisiae is caused by a rapid increase in the medium pH. Three factors have been identified as being important in regulating the rise in medium pH: 1) the cell density, 2) the potassium acetate concentration of the sporulation medium, and 3) and initial pH below 6.0. Sporulation conditions were established for strain 4579 which resulted in optimum uptake of ³H-adenine at T₇, a period when the cells would be normally impermeable. Pulse-labeled polysomal RNA was characterized at T₄ in naturally permeable cells of strain SK-1 and impermeable cells which required manipulation of the medium pH to facilitate uptake. Transfer ribonucleic acid (RNA), poly A-containing RNA and ribosomal RNA were synthesized in both cultures during the 20 min pulse. Furthermore, the rate of ribosomal RNA synthesis and processing into functional ribosomes approached the rate reported for vegetative cells. Initial sporulation conditions which caused a prolonged delay in the rise in medium pH adversely affected the kinetics of appearance and number of ascospores. The affect was shown to be on meiotic events since a reduction of sporulation was always accompanied by a reduction in the amount of intragenic recombination.