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.     

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.     

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.     

Mutants of Escherichia coli with an Altered Tryptophanyl-Transfer Ribonucleic Acid Synthetase

Fourteen mutant strains of Escherichia coli were examined, each of which requires tryptophan for growth but is unaltered in any of the genes of the tryptophan biosynthetic operon. The genetic lesions responsible for tryptophan auxotrophy in these strains map between str and malA. Extracts of these strains have little or no ability to charge transfer ribonucleic acid (tRNA) with tryptophan. We found that several of the mutants produce tryptophanyl-tRNA synthetases which are more heat-labile than the enzyme of the parental wild-type strain. Of these heat-labile synthetases, at least one is protected against thermal inactivation by tryptophan, magnesium, and adenosine triphosphate. Two other labile synthetases which are not noticeably protected against heat inactivation by substrate have decreased affinity for tryptophan. On low levels of supplied tryptophan, these mutants exhibit markedly decreased growth rates but do not contain derepressed levels of the tryptophan biosynthetic enzymes. This suggests that the charging of tryptophan-specific tRNA is not involved in repression, a conclusion which is further substantiated by our finding that 5-methyltryptophan, a compound which represses the tryptophan operon, is not attached to tRNA by the tryptophanyl-tRNA synthetase of E. coli.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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 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.     

Isoleucine and Valine Metabolism in Escherichia coli K-12: Detection and Measurement of ilv-Specific Messenger Ribonucleic Acid

Ribonucleic acid-deoxyribonucleic acid (RNA-DNA) hybridization was employed for the determination of messenger RNA transcribed from the ilv gene cluster of Escherichia coli K-12. Strains with derepressed levels of the isoleucine and valine biosynthetic enzymes owing to linked or unlinked genetic lesions were found to exhibit ilv messenger RNA levels from 1.5- to 4-fold higher than did their isogenic parents. When grown under conditions that specifically repressed the synthesis of isoleucine- and valine-forming enzymes, most strains exhibited drastically reduced ilv messenger RNA levels. Hybridization performed with the separated strands of ilv DNA showed that all the ilv genes are transcribed from the same strand, the "l strand" of lambdaphi80CI857St68dilv DNA. Sucrose gradient analyses of RNA extracted from cells starved for isoleucine, valine, or leucine resulted in the detection of at least two distinct types of ilv messenger RNA.     

Mutants of Escherichia coli with Cold-Sensitive Deoxyribonucleic Acid Synthesis

Ten cold-sensitive mutants defective in deoxyribonucleic acid (DNA) synthesis at 20 C have been identified among 218 cold-sensitive mutants isolated from a mutagenized population of Escherichia coli K-12. Four of the ten mutant alleles, dna-339 dna-340, dna-341, and dna-342, cotransduce with serB(+) and hence may be dnaC mutants. Two of these, dna-340 and dna-341, are recessive to their wild-type allele. The gene product of their wild-type allele is trans acting. Complementation tests have demonstrated that dna-340 and dna-341 are in the same cistron. The mapping of the remaining six mutations is in progress. In an attempt to determine whether LW4 and LW21 were initiator mutants, cultures of these strains were starved of an essential amino acid at 37 C and then incubated at 15 C with the essential amino acid. The amount of DNA synthesis observed under these circumstances was insignificant. These data are consistent with the idea that LW4 and LW21 are initiator mutants. However, attempts to integratively suppress LW4 and LW21 with F' factors were unsuccessful. To resolve the question of whether or not LW4 and LW21 are initiator mutants, more specific tests and criteria are required. Cultures of LW4 and LW21 were toluene treated and used to measure in vitro DNA synthesis. If the cells were incubated either at 15 or 20 C before toluene treatment, they were capable of markedly less DNA synthesis than if preincubation had not occurred. The amount of in vitro DNA synthesis is directly proportional to the amount of DNA synthesis occurring during preincubation in vivo; i.e., more DNA synthesis is observed at 20 than at 15 C. The fact that the cold-sensitive mutants are unable to synthesize DNA when supplied with deoxyribonucleoside triphosphates, DNA precursors, is evidence they are not defective in precursor synthesis.     

Control of Arginine Biosynthesis in Escherichia coli: Characterization of Arginyl-Transfer Ribonucleic Acid Synthetase Mutants

The arginyl-transfer ribonucleic acid (Arg-tRNA) synthetase (EC 6.1.1.13, arginine: RNA ligase adenosine monophosphate) mutants, exhibiting nonrepressible synthesis of arginine by exogenous arginine, were employed in studies of several biochemical properties. Two of these mutants possessed Arg-tRNA synthetases with a reduced affinity for arginine, and this enzyme of another mutant had a reduced affinity for arginine-tRNA (tRNA^arg). The mutant possessing an Arg-tRNA synthetase with an altered K_m for tRNA^arg was found to have reduced in vivo aminoacylation of two of the five isoaccepting species of tRNA^arg and complete absence of aminoacylation of one of the isoaccepting species.