Temperature-Sensitive Mutation in Regulation of Ribonucleic Acid Synthesis in Escherichia coli

An Escherichia coli mutant dependent on exogenous transfer ribonucleic acid (RNA) for bulk RNA formation at 42 C has been isolated, starting from a parental strain permeable to RNA. In the absence of added transfer RNA at the high temperature, protein synthesis stopped, and the strain formed little if any ribosomal RNA.     

Ribonucleic Acid Regulation in Permeabilized Cells of Escherichia coli Capable of Ribonucleic Acid and Protein Synthesis

A cell permeabilization procedure is described that reduces viability less than 10% and does not significantly reduce the rates of ribonucleic acid and protein synthesis when appropriately supplemented. Permeabilization abolishes the normal stringent coupling of protein and ribonucleic acid synthesis.     

Regulation of Ribonucleic Acid Synthesis by Histidine and Methionine During Recovery of Escherichia coli from Magnesium Starvation

During magnesium starvation of Escherichia coli B, most of the ribosomes break down to low-molecular-weight components. When magnesium is restored to the medium, the cells recover. The rate of recovery can be increased greatly by supplementing the growth medium with a mixture of 21 amino acids. This increased rate of recovery is shown to be due to the effect of only two amino acids, histidine and methionine, which initially stimulate accumulation of cellular ribonucleic acid without increasing the rate of protein synthesis. In contrast, histidine and methionine supplementation to logarithmically growing E. coli B is not as effective in stimulating growth as is the complete amino acid mixture. Since cells recovering from magnesium starvation preferentially synthesize ribosomes, it is possible that histidine and methionine play a special role(s) in ribosomal ribonucleic acid synthesis or stability.     

Continued Expression of the Ribonucleic Acid Control Gene During Inhibition of Escherichia coli Ribonucleic Acid and Protein Synthesis

The effect of the ribonucleic acid (RNA) control (RC) gene on the biosynthesis of viral RNA has been examined in an RC(str) and an RC(rel) host infected with R17 RNA bacteriophage under conditions in which host RNA and protein synthesis were inhibited by the addition of rifampicin. Methionine and isoleucine starvation depressed viral RNA biosynthesis in an RC(str) host but not in an RC(rel) host. However, histidine starvation had little effect on viral RNA and protein synthesis in both RC(str) and RC(rel) cells, although it had a marked effect on host protein and RNA synthesis in an RC(str) host. Chloramphenicol relieved the effect of amino acid starvation on viral RNA synthesis in an RC(str) host. It is concluded that stringent control of viral RNA biosynthesis does not require the continued biosynthesis of the RC gene product (RNA or protein) and that a preformed RC gene product can regulate the biosynthesis of the exogenous RNA. It is suggested that the amino acid dependence of viral RNA biosynthesis is due to its obligatory coupling with the translation of the viral coat protein which lacks histidine. It may be inferred that the amino acid requirement of bacterial RNA is due to its coupling with the translation of a host-specific protein (other than the RC gene product) which requires a full complement of amino acids. Since chloramphenicol is known to permit ribosome movement in the absence of protein synthesis, it is suggested that ribosome movement along the nascent RNA chain is a sufficient condition for the continuation of RNA synthesis.     

Ribonucleic Acid Synthesis and Glutamate Excretion in Escherichia coli

Cultures of Escherichia coli excreted glutamate into the medium when protein synthesis was blocked in RC(rel) strains or when it was blocked with chloramphenicol in either RC(str) or RC(rel) strains. Both of these conditions resulted in continued ribonucleic acid (RNA) synthesis in the absence of protein synthesis. Glutamate was also excreted by both RC(str) and RC(rel) strains when RNA synthesis was inhibited by uracil starvation or by treatment with actinomycin D. It is proposed that, in each of these cases, glutamate excretion resulted from an increase in the permeability of the cell membrane.     

Inhibition of Ribonucleic Acid Synthesis by Nalidixic Acid in Escherichia coli

The effect of low concentrations of nalidixic acid on ribonucleic acid (RNA) synthesis in Escherichia coli was examined. It was observed that RNA synthesis in exponentially growing cells was not significantly affected, in harmony with previous studies. However, RNA synthesis was markedly depressed by nalidixic acid during starvation for an amino acid or during chloramphenicol treatment. This effect was not caused by increased killing or inhibition of nucleoside triphosphate synthesis by nalidixic acid. The pattern of radioactive uracil incorporation into transfer RNA or ribosomes was not changed by the drug. The sensitivity of RNA synthesis to nalidixic acid in the absence of protein production may be useful in probing the amino acid control of RNA synthesis.     

Ribosomal Precursors and Ribonucleic Acid Synthesis in Escherichia coli

Ribosomes and immature ribonucleoprotein particles were isolated from extracts of log-phase cells grown under various conditions. Quantitative measurements were made to determine the relative amounts of immature particles present in the extracts. The results indicate that the steady-state level of ribosomal precursors accounted for essentially a constant fraction of the total ribonucleic acid (RNA) of the cells. For cells with RNA-protein ratios between 0.43 and 0.65, about 1.6% of the total RNA occurred as immature ribonucleoprotein particles. Further, increased levels of immature particles were shown to be correlated with a reduced rate of RNA synthesis in cells recovering from chloramphenicol inhibition. The reduction was found to vary directly with the duration of pretreatment in chloramphenicol and, consequently, with the level of immature particles present in the cells.     

Control of Deoxyribonucleic Acid and Ribonucleic Acid Synthesis in Pyrimidine-Limited Escherichia coli

The effects of pyrimidine limitation on chromosome replication and the control of ribosomal and transfer ribonucleic acid syntheses were investigated. Chromosome replication was studied by autoradiography of (3)H-thymine pulse-labeled cells. Pyrimidine limitation did not affect the fraction of cells incorporating radioactive thymine during a short pulse, indicating that when growth is limited by the supply of pyrimidine, the time required for chromosome duplication increases in proportion to the time required for cell duplication. Control of ribosomal RNA and transfer RNA syntheses was examined by chromatographing cell extracts on methylated albumin kieselguhr columns. When growth was controlled by carbon-nitrogen limitation, the ratio of tRNA to total RNA remained roughly constant at growth rates above 0.5 doublings per hour. During pyrimidine limitation, however, the control of rRNA synthesis was apparently dissociated from the control of tRNA synthesis: the ratio of tRNA to total RNA increased as the growth rate decreased.     

Control of Nucleotide Metabolism and Ribosomal Ribonucleic Acid Synthesis During Nitrogen Starvation of Escherichia coli

Ribosomal ribonucleic acid (RNA) synthesis and ribonucleoside triphosphate metabolism were studied in cultures of Escherichia coli subjected to starvation for inorganic nitrogen. In a strain that was under stringent control, a 50-fold reduction in the formation of both 16S and 23S RNA was accompanied by a severe restriction on nucleotide biosynthesis. These inhibitions were relieved in part by incubating the starved cells with amino acids. This result suggests that regulation by the functional RNA control (RC) gene is involved in the effect. This suggestion was confirmed by showing that the effector of the stringent response, guanosine-5'-diphosphate-2'- or 3'-diphosphate ((pp)G(pp)), accumulated at the onset of starvation and disappeared immediately when the amino acids were added. Ribosomal RNA synthesis was severely restricted and the same nucleotide, (pp)G(pp), accumulated at the onset of nitrogen starvation of a relaxed mutant too. These findings suggest that a control mechanism other than the one provided by the functional rel gene might operate to regulate RNA synthesis and that this mechanism is expressed through the synthesis of (pp)G(pp).     

Chloramphenicol and the Stimulation of Ribonucleic Acid Synthesis in Escherichia coli

Data have been obtained which imply that chloramphenicol stimulation of ribonucleic acid (RNA) synthesis is a result of the accumulation of aminoacyl transfer RNA (tRNA) molecules. The data also support the hypothesis that chloramphenicol exerts an additional effect upon the stimulation of RNA synthesis. This effect may be at the level of the ribosome or the aminoacyl tRNA, or of both. It is this effect combined with the presence of aminoacyl tRNA that results in stimulation by chloramphenicol of RNA synthesis.     

Stimulation of Unbalanced Ribonucleic Acid Synthesis in Escherichia coli by Methanol

Data are presented which support the view that l-lysine is transported by two systems in Streptococcus faecalis. The system with the higher affinity for l-lysine appears to be specific for l-lysine among the common amino acids and to require an energy source. The second system transports both l-lysine and l-arginine and does not appear to require an energy source. Both of these systems will accept hydroxy-l-lysine as a substrate as shown by the energy requirement for hydroxy-l-lysine transport and by the inhibition of uptake by l-arginine as well as by l-lysine. The affinity of both systems appears to be considerably lower for hydroxy-l-lysine than for l-lysine. A mutant of S. faecalis which is resistant to the growth inhibitory action of hydroxy-l-lysine appears to differ from the parent strain by having a defective l-lysine-specific transport system. In this mutant, hydroxy-l-lysine is not readily transported via the l-lysine-specific system because of the mutation or via the second system because of the high concentration of l-arginine present in the growth medium. This overall lack of transport prevents hydroxy-l-lysine from reaching inhibitory levels within the cell.     

Regulation of Synthesis of Methionyl-, Prolyl-, and Threonyl-Transfer Ribonucleic Acid Synthetases of Escherichia coli

Proline- and threonine-restricted growth caused a three- to fourfold derepression of the differential rate of synthesis of the prolyl- and threonyl-transfer ribonucleic acid (tRNA) synthetases, respectively. Similarly, there was approximately a 24-fold derepression in the rate of synthesis of methionyl-tRNA synthetase during methionine restriction. Addition of the respective amino acids to such derepressed cultures resulted in a repression of synthesis of their cognate synthetases. These results support previous findings and further strengthen the idea that the formation of aminoacyl-tRNA synthetases is regulated by some mechanism which is mediated by the cognate amino acids.     

Regulation of Synthesis of the Aminoacyl-Transfer Ribonucleic Acid Synthetases for the Branched-Chain Amino Acids of Escherichia coli

The regulation of synthesis of valyl-, leucyl-, and isoleucyl-transfer ribonucleic acid (tRNA) synthetases was examined in strains of Escherichia coli and Salmonella typhimurium. When valine and isoleucine were limiting growth, the rate of formation of valyl-tRNA synthetase was derepressed about sixfold; addition of these amino acids caused repression of synthesis of this enzyme. The rate of synthesis of the isoleucyl- and leucyl-tRNA synthetases was derepressed only during growth restriction by the cognate amino acid. Restoration of the respective amino acid to these derepressed cultures caused repression of synthesis of the aminoacyl-tRNA synthetase, despite the resumption of the wild-type growth rate.     

Stimulation of Ribonucleic Acid Synthesis by Chloramphenicol in a rel+ Aminoacyl-Transfer Ribonucleic Acid Synthetase Mutant of Escherichia coli

Escherichia coli strain 9D3 possesses a highly temperature-sensitive valyl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.9). Since 9D3 is a rel(+) strain, it cannot carry out net RNA synthesis at high temperature. A 100-mug amount of chloramphenicol (CAP) per ml added in the absence of valine cannot stimulate RNA synthesis. Either 300 mug of CAP or 100 mug of CAP plus 50 mug of valine per ml, however, promotes nearly maximal RNA synthesis. These results can be understood as follows. (i) Valyl-tRNA is required for net RNA synthesis, (ii) the synthetase lesion is incomplete, (iii) the rate of mutant acylation of tRNA(val) at high temperature is valine-dependent, and (iv) the CAP concentration determines the rate of residual protein synthesis. Data are also presented which demonstrate that the rate of net RNA synthesis can greatly increase long after the addition of CAP, if the amount of valyl-tRNA increases.     

Ribonucleic Acid Synthesis During Morphogenesis in Myxococcus xanthus

Ribonucleic acid synthesis was measured during the morphogenesis of Myxococcus xanthus. After induction of microcyst formation by the addition of glycerol to an exponential culture, net ribonucleic acid (RNA) synthesis was immediately terminated (measured either chemically or by the accumulation of acid-insoluble radioactivity). Extensive RNA turnover did take place, however, including RNA made both before and after induction. Sucrose gradient centrifugation revealed that ribosomes and ribosomal RNA were synthesized during microcyst formation even though there was no net RNA synthesis. Base analyses of the total RNA of vegetative cells and 120-min microcysts were indistinguishable.     

Reversal of the Mannitol-Sorbitol Diauxie in Escherichia coli

In Escherichia coli K-12 the proteins involved in the dissimilation of mannitol and sorbitol are specified by two separate gene clusters. The mannitol cluster appears to consist of a regulatory gene mtlC, a gene mtlA coding an enzyme II complex of the phosphoenolpyruvate phosphotransferase system, and a gene mtlD coding a mannitol-1-phosphate dehydrogenase. Three corresponding genes, sblC, sblA, and sblD, exist for the sorbitol pathway. In both pathways the hexitol captured from the medium and delivered into the cytoplasm as a phosphorylated compound is dehydrogenated to fructose-6-phosphate. The enzyme II complex for sorbitol is able to catalyze the phosphorylation also of mannitol if this substrate is present at high concentrations. Consequently mtlA(-) mutants lacking the enzyme II complex for mannitol can grow on mannitol either if the sorbitol phosphorylating system is preinduced by sorbitol or if mtlA is suppressed by a mutation of sblC to constitutivity. In wild-type cells, the induction of the enzymes in the mannitol pathway and dissimilation of the substrate are not prevented by glucose. The sorbitol system, however, is sensitive to glucose and to mannitol as well. In the suppressed strains (mtlA(-), sblC(c)) in which mannitol is utilized through the sorbitol enzyme, glucose becomes effective in restraining the consumption of mannitol, causing a definite diauxie. Moreover, in a mixture of mannitol and sorbitol, the latter is utilized preferentially. This reversal of normal diauxic pattern is consequent to the fact that the enzyme II complex for sorbitol has relatively poor affinity for mannitol.     

Correlation Between the Rate of Ribonucleic Acid Synthesis and the Level of Valyl Transfer Ribonucleic Acid in Mutants of Escherichia coli

By use of a mutant of Escherichia coli with a partially thermolabile transfer ribonucleic acid (tRNA) synthase, it was possible to regulate the rate of RNA synthesis over a 10-fold range. The addition of chloramphenicol to cultures kept at the nonpermissive temperature stimulated RNA synthesis. The longer the culture was kept at the nonpermissive temperature prior to addition of chloramphenicol, the lower was the resulting rate of RNA synthesis. The decrease in the rate of incorporation of labeled uracil into RNA was correlated with the decrease in the level of valyl tRNA. Additional experiments provided evidence which may be interpreted as indicating that valyl tRNA does not, by itself, react with the RNA-forming system.