Growth-Linked Instability of a Mutant Valyl-Transfer Ribonucleic Acid Synthetase in Escherichia coli

The valyl-transfer ribonucleic acid (tRNA) synthetase of Escherichia coli strain NP2907, previously described as having an elevated K(m) for adenosine triphosphate and reduced stability in vitro compared to the wild type, was found to be conditionally thermolabile in vivo. The rate of inactivation of this enzyme at a particular temperature appears to be coordinated with the rate of growth; at 40 C this coordination results in equal rates of synthesis and destruction over a wide range of growth rates. In vitro studies showed that conditions favoring maintenance of the valyl-tRNA synthetase-valyl adenylate complex conferred complete protection against inactivation at 40 C, whereas the further addition of uncharged tRNA caused rapid, irreversible decay. We propose that the rate of inactivation of this mutant valyl-tRNA synthetase in vivo is a function of the ratio of deacylated to acylated tRNA(val) and that this ratio is a function of growth rate. The event which renders the valyl-tRNA synthetase susceptible to inactivation is likely to be the normal breakdown of the valyl-tRNA synthetase-valyl-adenylate complex during a cycle of aminoacylation of tRNA(val).     

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.     

Characterization of Mutants of Escherichia coli Temperature-Sensitive for Ribonucleic Acid Regulation: an Unusual Phenotype Associated with a Phenylalanyl Transfer Ribonucleic Acid Synthetase Mutant

A mutant strain AA-522, temperature-sensitive for protein synthesis, was isolated from a stringent strain (CP-78) of Escherichia coli K-12. The mutant strain has a relaxed phenotype at the nonpermissive growth temperature. Protein synthesis stops completely at 42 C, whereas the rate of ribonucleic acid (RNA) synthesis is maintained at 20% of the 30 C rate. Sucrose-gradient centrifugation analysis of RNA-containing particles formed at 42 C indicated the presence of “relaxed particles.” These particles possess 16S and 23S RNA and are precursors to normal 50S and 30S ribosomal subunits. A search for the temperature-sensitive protein responsible for the halt in protein synthesis implicated phenylalanyl transfer RNA (tRNA) synthetase. Essentially no enzyme activity is detected in vitro at 30 or 40 C. Analysis of phenylalanyl tRNA synthetase activity in revertants of strain AA-522 indicated the presence of intragenic suppressor mutations. Revertants of strain AA-522 analyzed for the relaxed response at 42 C were all stringent; strain AA-522 was stringent at 30 C. These data indicate that a single mutation in phenylalanyl tRNA synthetase is responsible for both a block in protein synthesis and the relaxed phenotype at 42 C.     

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.     

Isolation and Partial Characterization of a Temperature-Sensitive Escherichia coli Mutant with Altered Glutaminyl-Transfer Ribonucleic Acid Synthetase

A temperature-sensitive mutant of Escherichia coli has been found in which the conditional growth is a result of a thermosensitive glutaminyl-transfer ribonucleic acid synthetase. The corresponding genetic locus glnS is cotransduced with lip. In a strain containing the mutationally altered glutaminyl-transfer ribonucleic acid synthetase, no derepression of the enzyme itself nor of glutamine synthetase was observed.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Control of Arginine Biosynthesis in Escherichia coli: Role of Arginyl-Transfer Ribonucleic Acid Synthetase in Repression

The physiological role of arginyl-transfer ribonucleic acid (Arg-tRNA) synthetase (E.C. 6.1.1.13, arginine: RNA ligase adenosine monophosphate) in repression of arginine biosynthetic enzymes was examined. Mutants with nonrepressible synthesis of arginine biosynthetic enzymes were isolated from various strains of Escherichia coli by resistance to growth inhibition by canavanine, an arginine analogue. These mutants possessed reduced Arg-tRNA synthetase activities which were qualitatively different from the synthetase activity of the wild type. The mutant enzymes exhibited turnover in vivo and were less stable in vitro than the wild type at both 4 C and 40 C; they possessed different affinities for both arginine and canavanine as measured by the three common assay systems for aminoacyl-tRNA synthetases. Furthermore, in one case it was shown that (i) the mutant possesed unaltered uptake of arginine, and (ii) that the mutant possessed diminished ability to incorporate canavanine into proteins and to attach canavanine to tRNA. These observations suggested that the mutation to canavanine resistance involved a structural change in Arg-tRNA synthetase. Likewise, the results of genetic experiments suggested that the mutants differed from the wild-type strain at only one locus, and that this lies in the region of the chromosomes that includes a structural gene for Arg-tRNA synthetase. It appears that Arg-tRNA synthetase may be involved in some way in repression by arginine of its own biosynthetic enzymes.     

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.     

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.     

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.     

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.