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.     

Isolation and Characterization of a Regulatory Mutant of an Aminoacyl-Transfer Ribonucleic Acid Synthetase in Escherichia coli K-12

From Escherichia coli strain K28, which is temperature sensitive for growth because of a mutation in its seryl-transfer ribonucleic acid (tRNA) synthetase gene (serS), temperature-resistant mutants were selected which were found to have a fivefold higher level of seryl-tRNA synthetase than the parent strain. The "high-level" character was found to be genetically stable and is due to a mutation in a locus denoted serO. This locus was found to be very closely linked to serS on the genetic map, and the relative gene order was concluded to be serS-serO-serC. In a serO(-) strain, the normal dependence of seryl-tRNA synthetase (SerRS) activity on changes of exogenous serine concentration was not observed. In a stable heterozygous merodiploid, the serO(-) mutation is still expressed, i.e., it is cis dominant. These results strongly suggest that serO is an operator site involved in the control of the serS gene.     

Unusual Valyl-Transfer Ribonucleic Acid Synthetase Mutant of Escherichia coli

Escherichia coli strain NP2907 was isolated as a spontaneous mutant of strain NP29, which possesses a thermolabile valyl-transfer ribonucleic acid (tRNA) synthetase. The valyl-tRNA synthetase of the new mutant, unlike that of its immediate parent, retains enzymatic activity in vitro but differs from the wild-type enzyme in stability and apparent K(m) for adenosine triphosphate. The new mutant locus, valS-102, cotransduces with pyrB at the same frequency as does the parental locus, valS-1. Cultures of strain NP29 cease growth immediately in any medium when shifted from 30 to 40 C. The new mutant grows normally at 30 C, and upon a shift to 40 C growth quickly accelerates exactly as for normal cells. Exponential growth, however, cannot be sustained at 40 C. At a point characteristic for each medium, growth becomes linear with time. This transition occurs almost immediately in rich media and after 1.5 generations in glucose minimal medium. Net synthesis of valyl-tRNA synthetase ceases in the new mutant as soon as the temperature is raised to 40 C, irrespective of the growth medium. We conclude that it is the amount of valyl-tRNA synthetase activity that limits the rate of growth in the linear phase at 40 C. This property of the mutant makes it possible to evaluate the in vivo efficiency of this enzyme at different growth rates and thereby to determine the concentration that is necessary for a given rate of protein synthesis. The results of our measurements indicate that cells of E. coli growing in minimal medium normally possess a functional excess of valyl-tRNA synthetase with respect to protein synthesis and to repression of threonine deaminase.     

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.     

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

The kinetics of derepression of valyl-, isoleucyl-, and leucyl-transfer ribonucleic acid (tRNA) synthetase formation was examined during valine-, isoleucine-, and leucine-limited growth. When valine was limiting growth, valyl-tRNA synthetase formation was maximally derepressed within 5 min, whereas the rates of synthesis of isoleucyl-, and leucyl-tRNA synthetases were unchanged. Isoleucine-restricted growth caused a maximal derepression of isoleucyl-tRNA synthetase formation in 5 min and derepression of valyl-tRNA synthetase formation in 15 min with no effect on leucyl-tRNA synthetase formation. When leucine was limiting growth, leucyl-tRNA synthetase formation was immediately derepressed, whereas valyl- and isoleucyl-tRNA synthetase formation was unaffected by manipulation of the leucine supply to the cells. These results support our previous findings that valyl-tRNA synthetase formation is subject to multivalent repression control by both isoleucine and valine. In contrast, repression control of iso-leucyl- and leucyl-tRNA synthetase formation is specifically mediated by the supply of the cognate amino acid.     

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.     

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.     

Mapping of a Structural Gene for Valyl-Transfer Ribonucleic Acid Synthetase in Escherichia coli by Transduction

A structural gene, valS, for the valyl-transfer ribonucleic acid synthetase of Escherichia coli has been mapped on the clockwise side of pyrB and is closely linked to it.     

Neurospora Mutant Deficient in Tryptophanyl-Transfer Ribonucleic Acid Synthetase Activity

A tryptophan auxotroph of Neurospora crassa, trp-5, has been characterized as a mutant with a deficient tryptophanyl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.2) activity. When assayed by tryptophanyl-tRNA formation, extracts of the mutant have less than 5% of the wild-type specific activity. The adenosine triphosphate-pyrophosphate exchange activity is at about half the normal level. In the mutant derepressed levels of anthranilate synthetase and tryptophan synthetase were associated with free tryptophan pools equal to or higher than those found in the wild type. We conclude that a product of the normal tryptophanyl-tRNA synthetase, probably tryptophanyl-tRNA, rather than free tryptophan, participates in the repression of the tryptophan biosynthetic enzymes.     

Ribonucleic Acid Polymerase Mutant of Escherichia coli Defective in Flagella Formation

Escherichia coli K-12 mutants that are resistant to bacteriophage chi, defective in motility, and unable to grow at high temperature (42 degrees C) were isolated from among those selected for rifampin resistance at low temperature (30 degrees C) after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Genetic analysis of one such mutant indicated the presence of two mutations that probably affect the beta subunit of ribonucleic acid (RNA) polymerase: one (rif) causing rifampin resistance and the other (Ts-74) conferring resistance to phage chi (and loss of motility) and temperature sensitivity for growth. Observations with an electron microscope revealed that the number of flagella per mutant cell was significantly reduced, suggesting that the Ts-74 mutation somehow affected flagella formation at the permissive temperature. When a mutant culture was transferred from 30 to 42 degrees C, deoxyribonucleic acid synthesis accelerated normally, but RNA or protein synthesis was enhanced relatively little. The rate of synthesis of beta and beta' subunits of RNA polymerase was low even at 30 degrees C and was further reduced at 42 degrees C, in contrast to the parental wild-type strain. Expression of the lactose and other sugar fermentation operons, as well as lysogenization with phage lambda, occurred normally at 30 degrees C, suggesting that the mutation does not cause general shut-off of gene expression regulated by cyclic adenosine 3',5'-monophosphate.     

Inhibition of Arginyl-Transfer Ribonucleic Acid Synthetase Activity of Escherichia coli by Arginine Biosynthetic Precursors

The arginine biosynthetic precursors, ornithine, citrulline, and argininosuccinate, inhibit arginyl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.13, arginine: soluble RNA ligase, adenosine monophosphate) activity in the in vitro attachment assay system. Ornithine is the most potent, argininosuccinate is next, and citrulline is least effective. The implications of these results are discussed in relation to arginyl-tRNA synthetase activity and the level of the arginine biosynthetic enzymes during conditions of restricted and unrestricted supply of arginine to cells.     

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.     

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.     

Mutants of Escherichia coli K-12 with an Altered Glutamyl-Transfer Ribonucleic Acid Synthetase

Three streptomycin-suppressible lethal mutants of Escherichia coli K-12 have been shown to possess structurally altered glutamyl-transfer ribonucleic acid (tRNA) synthetases. Each mutant synthetase displays a K(m) value for glutamate which is 10-fold higher than the parental value, and the mutations reside in two widely separate loci on the genetic map. Mixing of the mutant extracts in pairs gave no indication of in vitro complementation. All three enzymes charge the minor tRNA(glu) fraction identically, but one (EM 120) charges the major fraction at a twofold lower rate than do the other two (EM 102 and EM 111). Possible explanations for the existence of the two synthetase loci are presented.     

Evidence for the Existence of Two Arginyl-Transfer Ribonucleic Acid Synthetase Activities in Escherichia coli

Two arginyl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.13, arginine: ribonucleic acid ligase adenosine monophosphate) activities were found in extracts of Escherichia coli strains AB1132 and NP2. The two arginyl-tRNA synthetase activities in extracts of strain AB1132 were found to be separable by diethylaminoethyl-cellulose column chromatography, Sephadex column fractionation, and by sucrose density gradient centrifugation. In addition, in the standard assay using extracts of strain AB1132 there were two pH optima for arginyl-tRNA synthetase activity. Furthermore, when arginyl-tRNA synthetase of strain NP2 was fractionated by hydroxylapatite column chromatography, two activities were observed which were similar to those of strain AB1132.     

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.     

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.     

Role of Isoleucyl-Transfer Ribonucleic Acid Synthetase in Ribonucleic Acid Synthesis and Enzyme Repression in Yeast

Temperature-sensitive mutations in the isoleucyl-transfer ribonucleic acid (tRNA) synthetase of yeast, ilS(-)1-1 and ilS(-)1-2, were used to examine the role of aminoacyl-tRNA synthetase enzymes in the regulation of ribonucleic acid (RNA) synthesis and enzyme synthesis in a eucaryotic organism. At the permissive temperature, 70 to 100% of the intracellular isoleucyl-tRNA was charged in mutants carrying these mutations; at growth-limiting temperatures, less than 10% was charged with isoleucine. Other aminoacyl-tRNA molecules remained essentially fully charged under both conditions. Net protein and RNA syntheses were rapidly inhibited when the mutant was shifted from the permissive to the restrictive temperature. Most of the ribosomes remained in polyribosome structures at the restrictive temperature even though protein synthesis was strongly inhibited. Two of the enzymes of isoleucine biosynthesis, threonine deaminase and acetohydroxyacid synthetase, were derepressed about twofold during slow growth of the mutants at a growth-limiting temperature. This is about the same degree of derepression that is achieved by growth of an auxotroph on limiting isoleucine. We conclude that charged aminoacyl-tRNA is essential for RNA synthesis and for the multivalent repression of the isoleucine biosynthetic enzymes. Aminoacyl tRNA synthetase enzymes appear to play important regulatory roles in the cell physiology of eucaryotic organisms.     

Control of Arginine Biosynthesis in Escherichia coli: Inhibition of Arginyl-Transfer Ribonucleic Acid Synthetase Activity

In this study, we have extended our earlier observations indicating in vitro inhibition of arginyl-transfer ribonucleic acid synthetase (EC 6.1.13, arginine: soluble ribonucleic acid ligase, adenosine monophosphate) activity by the arginine biosynthetic precursors ornithine, citrulline, and argininosuccinate. Furthermore, we report evidence which suggest that this enzyme activity is inhibited by these arginine precursors in vivo and that this inhibition of activity results in a derepression of arginine biosynthesis.