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 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.     

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.     

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.     

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.     

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.     

Regulation of Ribonucleic Acid Synthesis in Escherichia coli During Diauxie Lag: Accumulation of Heterogeneous Ribonucleic Acid

The synthesis of ribonucleic acid (RNA) and of protein in Escherichia coli during glucose-lactose diauxie lag have been examined. The rate of RNA synthesis is about 7%, of the corresponding rate during exponential growth and the rate of protein synthesis 10 to 15%. Inhibition of RNA synthesis occurs to the same extent in both rel and rel(+) strains. The RNA which accumulates during 20 min in diauxie lag is composed of about 50% ribosomal and transfer RNA species and about 50% of a fraction which resembles messenger RNA (mRNA) in its heterogeneous sedimentation properties. Decay of the heterogeneous fraction occurs in the presence of glucose and actinomycin D with a half-life of 3 min, the same as that of pulse-labeled mRNA; however, during the diauxie lag, the half-life of this RNA is about 25 min. Accumulation of the heterogeneous RNA is further increased when protein synthesis is blocked by chloramphenicol. The data suggest that the disproportionate accumulation of mRNA during diauxie lag and energy source shift-down may be attributed at least in part to increased stability of mRNA, but do not rule out a preferential synthesis of mRNA.     

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.     

Messenger Ribonucleic Acid Synthesis and Degradation in Escherichia coli During Inhibition of Translation

Various aspects of the coupling between the movement of ribosomes along messenger ribonucleic acids (mRNA) and the synthesis and degradation of mRNA have been investigated. Decreasing the rate of movement of ribosomes along an mRNA does not affect the rate of movement of some, and possibly most, of the RNA polymerases transcribing the gene coding for that mRNA. Inhibiting translation with antibiotics such as chloramphenicol, tetracycline, or fusidic acid protects extant mRNA from degradation, presumably by immobilizing ribosomes, whereas puromycin exposes mRNA to more rapid degradation than normal. The promoter distal (3') portion of mRNA, synthesized after ribosomes have been immobilized by chloramphenicol on the promoter proximal (5') portion of the mRNA, is subsequently degraded.     

Putrescine-mediated Degradation of Ribonucleic Acid in Chloramphenicol-treated Escherichia coli

Escherichia coli treated with chloramphenicol (CM) accumulated ribonucleic acid (RNA) in the absence of protein synthesis. The accumulated RNA (CM-RNA) was largely ribosomal (23S and 16S) and soluble (4S). The stability of CM-RNA depended upon the incubation conditions following the removal of CM. Thus, conditions which allowed the complete recovery of cultures from CM inhibition resulted in only a 30% loss of CM-RNA. The addition of proflavine to recovering cultures, which prevented further RNA synthesis, also resulted in about 30 to 35% degradation of CM-RNA. However, when RNA synthesis was inhibited by starving the recovering cultures for the required amino acid, histidine, 55% of the CM-RNA was degraded. The decreased stability of CM-RNA in histidine-starved cultures appeared to be due specifically to the intracellular buildup of putrescine. Under the above conditions of incubation, that RNA which was stable sedimented in sucrose gradients as 23S, 16S, and 4S RNA. It is suggested that intracellular putrescine plays a role in the stability of ribosomal RNA accumulated during CM treatment.     

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.     

Synthesis of Ribonucleic Acid and Protein in Plasmid-Containing Minicells of Escherichia coli K-12

Unlike the deoxyribonucleic acid (DNA)-deficient minicells produced by F(-) parents, minicells produced by plasmid-containing strains contain significant amounts of plasmid DNA. We examined the ability of plasmid-containing minicells to synthesize ribonucleic acid (RNA) and protein. In vivo, minicells produced by F(-) parents are unable to incorporate radioactive precursors into acid-insoluble RNA or protein, whereas minicells produced by F', R(+), or Col(+) parents are capable of such synthesis. Using a variety of approaches, including polyacrylamide gel analysis of the RNA species produced and electron microscope autoradiography, we demonstrated that the synthesis observed in minicell preparations is a property of the plasmid-containing minicells and not a result of the few cells (approximately 1 per 10(6) minicells) contaminating the preparations. That the observed synthesis is of biological importance is suggested by the ability of plasmid-containing minicells to yield viable phage upon infection with T4.     

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.     

Host-Virus Interaction in Ribonucleic Acid Bacteriophage-Infected Escherichia coli: I. Location of “Late” MS2-Specific Ribonucleic Acid Synthesis

When actinomycin-treated, MS2-infected Escherichia coli are labeled during a brief period later than 16 min after infection, the newly synthesized MS2 ribonucleic acid (RNA) appears first in the 30,000 x g sediment, probably bound to fragments of bacterial membranes, since the radioactivity can be released from the sediment with deoxycholate or urea. With longer labeling times, radioactivity also appears in the 30,000 x g supernatant fluid. While on the membrane, the RNA is organized into particles with sedimentation coefficients of 40, 32, and 27S in the presence of low Mg(2+). In the presence of high Mg(+), MS2-specific RNA is found in polyribosomes. These data are interpreted to mean that MS2-specific RNA is synthesized and organized into larger structures on membrane. More than 8 min of labeling is required before radioactivity is found in the 81S virion which appears in the supernatant fluid.     

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.