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.     

Close linkage of the genes serC (for phosphohydroxy pyruvate transaminase) and serS (for seryl-transfer ribonucleic acid synthetase) in Escherichia coli K-12

Escherichia coli strain K28, isolated after nitrosoguanidine mutagenesis, was found to be auxotrophic for serine. It was also temperature sensitive for growth as a result of producing an altered seryl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.11, l-serine: tRNA ligase [AMP]). The auxotrophy was caused by a mutation in the structural gene for phosphohydroxy-pyruvate transaminase (serC), which was distinct from, but closely linked to, the structural gene for seryl-tRNA synthetase (serS). We conclude that the relevant genes are in the order gal-serS-serC-aroA.     

Cloning and characterization of the gene coding for cytoplasmic seryl-tRNA synthetase from Saccharomyces cerevisiae.

We have screened a Saccharomyces cerevisiae expression library with antibodies against seryl-tRNA synthetase (SerRS) from baker's yeast. In this way we obtained clones which contain serS, the structural gene for seryl-tRNA synthetase. Genomic Southern blots show that the serS gene resides on a 5.0 kb SalI fragment. Nucleotide sequence analysis of the genes revealed a single open reading frame from which we deduced the amino acid sequence of the enzyme consistent with that of two peptides isolated from SerRS. The enzyme is comprised of 462 amino acids consistent with earlier determinations of its molecular weight. The codon usage of serS is typical of abundant yeast proteins. Nuclease S1 analysis of serS mRNA defined the RNA initiation site 20-40 bases downstream from an AT rich sequence containing the TATA box and 21-39 nucleotides upstream of the translation initiation codon. Yeast strains transformed with the cloned gene overproduce seryl-tRNA synthetase in vivo.     

Cloning and characterization of the gene for Escherichia coli seryl-tRNA synthetase.

Seryl-tRNA synthetase is the gene product of the serS locus in Escherichia coli. Its gene has been cloned by complementation of a serS temperature sensitive mutant K28 with an E. coli gene bank DNA. The resulting clones overexpress seryl-tRNA synthetase by a factor greater than 50 and more than 6% of the total cellular protein corresponds to the enzyme. The DNA sequence of the complete coding region and the 5'- and 3' untranslated regions was determined. Protein sequence comparison of SerRS with all available aminoacyl-tRNA synthetase sequences revealed some regions of significant homology particularly with the isoleucyl- and phenylalanyl-tRNA synthetases from E. coli.     

Thymidine 5′-Monophosphate-Requiring Mutants of Saccharomyces cerevisiae Are Deficient in Thymidylate Synthetase

Thymidylate synthetase activity was measured in crude extracts of the yeast Saccharomyces cerevisiae by a sensitive radiochemical assay. Spontaneous non-conditional mutants auxotrophic for thymidine 5'-monophosphate (tmp1) lacked detectable thymidylate synthetase activity in cell-free extracts. In contrast, the parent strains (tup1, -2, or -4), which were permeable to thymidine 5'-monophosphate, contained levels of activity similar to those found in wild-type cells. Specific activity of thymidylate synthetase in crude extracts of normal cells or of cells carrying tup mutations was essentially unaffected by the ploidy or mating type of the cells, by the medium used for growth, by the respiratory capacity of the cells, by concentrations of exogenous thymidine 5'-monophosphate as high as 50 mug/ml, or by subsequent removal of thymidine 5'-monophosphate from the medium. Extracts of a strain bearing the temperature-sensitive cell division cycle mutation cdc21 lacked detectable thymidylate synthetase activity under all conditions tested. Its parent and another mutant (cdc8), which arrests with the same terminal phenotype under restrictive conditions, had normal levels of the enzyme. Cells of a temperature-sensitive thymidine 5'-monophosphate auxotroph arrested with a morphology identical to the cdc21 strain at the nonpermissive temperature and contained demonstrably thermolabile thymidylate synthetase activity. Tetrad analysis and the properties of revertants showed that the thymidylate synthetase defects were a consequence of the same mutation causing, in the auxotrophs, a requirement for thymidine 5'-monophosphate and, in the conditional mutants, temperature sensitivity. Complementation tests indicated that tmp1 and cdc21 are the same locus. These results identify tmp1 as the structural gene for yeast thymidylate synthetase.     

Reversion of the effects of a threonine deaminase regulatory mutant by a mutation in ilvH in Escherichia coli K-12

In a strain carrying an ilvA538 mutation, the ilvGEDA operon expression is decreased (hyperattenuated) and the activity and/or expression of isoleucyl- and valyl- tRNA synthetases is decreased. We have isolated two revertants of ilvA538 owing to mutations in the ilvH gene, whose product is acetohydroxy acid synthase III. The regulatory properties of these revertants are consistent with a dual role for threonine deaminase as an effector of the ilvGEDA operon and the isoleucyl- and valyl- tRNA synthetase structural genes.     

Conserved polar loop region of Escherichia coli subunit c of the F1F0 H+-ATPase. Glutamine 42 is not absolutely essential, but substitutions alter binding and coupling of F1 to F0

The uncE114 mutation (Gln42----Glu) in subunit c of the Escherichia coli H+ ATP synthetase causes uncoupling of proton translocation from ATP hydrolysis (Mosher, M. E., White, L. K., Hermolin, J., and Fillingame, R. H. (1985) J. Biol. Chem. 260, 4807-4814). In the background of strain ER, the mutation led to dissociation of F1 from the membrane. Ten revertants to the uncE114 mutation were isolated, and the uncE gene was cloned and sequenced. Six of the revertants were intragenic and had substitutions of glycine, alanine, or valine for the mutant glutamate residue at position 42. The intragenic, revertant uncE genes were incorporated into an otherwise wild type chromosome of strain ER. Membrane vesicles prepared from each of the revertants showed a restoration of F1 binding to F0. The Val42 revertant differed from the other two revertants in that the ATPase activity of F1 was inhibited when membrane bound. This was shown by the stimulation of ATPase activity when F1 was released from the membrane. The Gly42 and Ala42 revertants demonstrated membrane ATPase activity that was resistant to dicyclohexylcarbodiimide treatment. Resistance was shown to be due to the increased dissociation of F1 from the membrane under ATPase assay conditions. The Ala42 revertant showed a significant reduction in ATP-dependent quenching of quinacrine fluorescence that was attributed to less efficient coupling of ATP hydrolysis to H+ translocation, whereas the other revertants showed responses very near to that of wild type. Minor changes in the F1-F0 interaction in all three revertants were indicated by an increase in H+ leakiness, as judged by reduced NADH-dependent quenching of quinacrine fluorescence. The minor defects in the revertants support the idea that residue 42 is involved in the binding and coupling of F1 to F0 but also show that the conserved glutamine (or asparagine) is not absolutely necessary in this function.     

Regulation of the biosynthesis of aminoacyl-tRNA synthetases and of tRNA in Escherichia coli. IV. Mutants with increased levels of leucyl- or seryl-tRNA synthetase.

Summary Spontaneous revertants of a temperature-sensitive Escherichia coli strain harboring a thermolabile leucyl-tRNA synthetase and seryl-tRNA synthetase were selected for growth at 40°C. Among these, strains were found with increased levels of both thermolabile synthetases. Two distinct genetic loci were found responsible for enzyme overproduction. leuR, located near xyl, causes elevated levels of leucyl-tRNA synthetase; while serR, located near leu, causes elevated levels of seryl-tRNA synthetase.The preceding paper in this series is by R. LaRossa, J. Mao, K.B. Low and D. Söll. J. Mol. Biol. 117, 1049 (1977)     

prsB is an allele of the Salmonella typhimurium prsA gene: characterization of a mutant phosphoribosylpyrophosphate synthetase.

The Salmonella typhimurium prsB mutation was previously mapped at 45 min on the chromosome, and a prsB strain was reported to produce undetectable levels of phosphoribosylpyrophosphate (PRPP) synthetase activity and very low levels of immunologically cross-reactive protein in vitro (N.K. Pandey and R.L. Switzer, J. Gen. Microbiol, 128:1863-1871, 1982). We have shown by P22-mediated transduction that the prsB gene is actually an allele of prsA, the structural gene for PRPP synthetase, which maps at 35 min. The prsB (renamed prs-100) mutant produces about 20% of the activity and 100% of the cross-reactive material of wild-type strains. prs-100 mutant strains are temperature sensitive, as is the mutant PRPP synthetase in vitro. The prs-100 mutation is a C-to-T transition which results in replacement of Arg-78 in the mature wild-type enzyme by Cys. The mutant PRPP synthetase was purified to greater than 98% purity. It possessed elevated Michaelis constants for both ATP and ribose-5-phosphate, a reduced maximal velocity, and reduced sensitivity to the allosteric inhibitor ADP. The mutant enzyme had altered physical properties and was susceptible to specific cleavage at the Arg-101-to-Ser-102 bond in vivo. It appears that the mutation alters the enzyme's kinetic properties through substantial structural alterations rather than by specific perturbation of substrate binding or catalysis.     

Biogenesis of mitochondria: a mutation in the 5''-untranslated region of yeast mitochondrial oli1 mRNA leading to impairment in translation of subunit 9 of the mitochondrial ATPase complex.

A temperature-conditional mit- mutant of Saccharomyces cerevisiae has been characterized; the mutant strain h45 cannot grow at 36 degrees C on nonfermentable substrates yet appears to be normal at 28 degrees C. The mutation in strain h45 maps genetically to the oli1 region of the mitochondrial DNA (mtDNA) genome, and prevents the synthesis at 36 degrees C of the oli1 gene product, subunit 9 of the mitochondrial ATPase complex. Since the level of oli1 mRNA in mutant h45 is close to normal at 36 degrees C, it is concluded that there is a specific block in translation of this mRNA at the non-permissive temperature. DNA sequence analysis of mtDNA from strain h45 reveals an additional T residue inserted 88 bp upstream of the oli1 coding region, in the A,T-rich sequence that is transcribed into the 5'-untranslated region of the oli1 mRNA. Sequence data on two revertants show that one returns to wild-type parental (J69-1B) mtDNA sequence, whilst the other contains an inserted A residue adjacent to the T inserted in the original h45 mutant. The results are discussed in terms of the stability of folds in RNA upstream of putative ribosome-binding sites in mitochondrial mRNA, and the potential action of nuclear-coded proteins that might be activators of the translation of specific mitochondrial mRNAs in yeast mitochondria.     

Altered regulation of the glnRA operon in a Bacillus subtilis mutant that produces methionine sulfoximine-tolerant glutamine synthetase.

A Bacillus subtilis mutant that produced glutamine synthetase (GS) with altered sensitivity to DL-methionine sulfoximine was isolated. The mutation, designated glnA33, was due to a T.A-to-C.G transition, changing valine to alanine at codon 190 within the active-site C domain. Altered regulation was observed for GS activity and antigen and mRNA levels in a B. subtilis glnA33 strain. The mutant enzyme was 28-fold less sensitive to DL-methionine sulfoximine and had a 13.0-fold-higher Km for hydroxylamine and a 4.8-fold-higher Km for glutamate than wild-type GS did.     

The isolation and characterization of glutamine-requiring strains of Escherichia coli K12.

Mutants of Escherichia coli K12 requiring glutamine as a nitrogen source were isolated, and characterized as lacking glutamine synthetase activity. Temperature sensitive revertants of one of the mutants had a heat labile glutamine synthetase, while temperature insensitive revertants had a glutamine synthetase which was thermostable in vitro, indicating that the mutation was in the structural gene for the enzyme. All of the mutations mapped in the same region of the chromosome suggesting that they might all be in the same gene. The glutamine synthetase gene (gln) was located on the E. coli chromosome by conjugation and P1-mediated transduction at minute 77. The gln gene cotransduced with the genes for oleate degradation (old), and the genes for L-rhamnose utilization (rha). The most probable gene order is old-gln-rha.     

Expression of interferon-induced genes in different tissues of mice.

In vivo responses to interferon (IFN) in mice were determined by measuring the steady-state levels of induced mRNAs following injection of IFN and poly(I)-poly(C). With cDNA probes for mouse 2'-5' oligoadenylate synthetase (2-5A synthetase) and 1-8, constitutive expression of the corresponding mRNA was detectable in different organs of normal C3H/He mice. These mRNA levels were increased by as much as 15-fold over control levels in various tissues, including the brain, after IFN and poly(I)-poly(C) treatment, coincident with increases in 2-5A synthetase enzyme activity. The basal activity level of this enzyme could be reduced in normal mice by treatment with anti-mouse IFN (alpha + beta) antibody. This treatment also reduced the levels of 2-5A synthetase and 1-8 mRNAs. Thus, physiological levels of circulating IFN maintain elevated levels of IFN-induced mRNAs in mice. Furthermore, changes in 2-5A synthetase enzyme activity reflect the changes in gene expression in vivo.     

The tRNA-dependent activation of arginine by arginyl-tRNA synthetase requires inter-domain communication

The tRNA-dependent amino acid activation catalyzed by mammalian arginyl-tRNA synthetase has been characterized. A conditional lethal mutant of Chinese hamster ovary cells that exhibits reduced arginyl-tRNA synthetase activity (Arg-1), and two of its derived revertants (Arg-1R4 and Arg-1R5) were analyzed at the structural and functional levels. A single nucleotide change, resulting in a Cys to Tyr substitution at position 599 of arginyl-tRNA synthetase, is responsible for the defective phenotype of the thermosensitive and arginine hyper-auxotroph Arg-1 cell line. The two revertants have a single additional mutation resulting in a Met222 to Ile change for Arg-1R4 or a Tyr506 to Ser change for Arg-1R5. The corresponding mutant enzymes were expressed in yeast and purified. The Cys599 to Tyr mutation affects both the thermal stability of arginyl-tRNA synthetase and the kinetic parameters for arginine in the ATP-PP(i) exchange and tRNA aminoacylation reactions. This mutation is located underneath the floor of the Rossmann fold catalytic domain characteristic of class 1 aminoacyl-tRNA synthetases, near the end of a long helix belonging to the alpha-helix bundle C-terminal domain distinctive of class 1a synthetases. For the Met222 to Ile revertant, there is very little effect of the mutation on the interaction of arginyl-tRNA synthetase with either of its substrates. However, this mutation increases the thermal stability of arginyl-tRNA synthetase, thereby leading to reversion of the thermosensitive phenotype by increasing the steady-state level of the enzyme in vivo. In contrast, for the Arg-1R5 cell line, reversion of the phenotype is due to an increased catalytic efficiency of the C599Y/Y506S double mutant as compared to the initial C599Y enzyme. In light of the location of the mutations in the 3D structure of the enzyme modeled using the crystal structure of the closely related yeast arginyl-tRNA synthetase, the kinetic analysis of these mutants suggests that the obligatory tRNA-induced activation of the catalytic site of arginyl-tRNA synthetase involves interdomain signal transduction via the long helices that build the tRNA-binding domain of the enzyme and link the site of interaction of the anticodon domain of tRNA to the floor of the active site.     

SAM2 encodes the second methionine S-adenosyl transferase in Saccharomyces cerevisiae: physiology and regulation of both enzymes.

In Saccharomyces cerevisiae the SAM1 and SAM2 genes encode two distinct forms of S-adenosylmethionine (AdoMet) synthetase. In a previous study we cloned and sequenced the SAM1 gene (D. Thomas and Y. Surdin-Kerjan, J. Biol. Chem. 262:16704-16709, 1987). In this work, the SAM2 gene was isolated by functional complementation of a yeast double-mutant strain, and its identity was ascertained by gene disruption. It has been sequenced and compared with the SAM1 gene. The degree of homology found between the two genes shows that SAM1 and SAM2 are duplicated genes. Using strains disrupted in one or the other SAM gene, we have studied the regulation of their expression by measuring the steady-state level of mRNA after growth under different conditions. The results show that the expression of the two SAM genes is regulated differently, SAM2 being induced by the presence of excess methionine in the growth medium and SAM1 being repressed under the same conditions. The level of mRNA in the parental strain shows that it is not the sum of the levels found in the two disrupted strains. This raises the question of how the two AdoMet synthetases in S. cerevisiae interact to control AdoMet synthesis.     

Effect of dibutyryl cyclic AMP and dexamethasone on glutamine synthetase gene expression in rat astrocytes in culture

Astrocytes are the primary site of glutamate conversion to glutamine in the brain. We examined the effects of treatment with either dibutyryl cyclic AMP and/or the synthetic glucocorticoid dexamethasone on glutamine synthetase enzyme activity and steady-state mRNA levels in cultured neonatal rat astrocytes. Treatment of cultures with dibutyryl cyclic AMP alone (0.25 mM–1.0 mM) increased glutamine synthetase activity and steady state mRNA levels in a dose-dependent manner. Similarly, treatment with dexamethasone alone (10^–7–10^–5 M) increased glutamine synthetase mRNA levels and enzyme activity. When astrocytes were treated with both effectors, additive increases in glutamine synthetase activity and mRNA were obtained. However, the additive effects were observed only when the effect of dibutyryl cyclic AMP alone was not maximal. These findings suggest that the actions of these effectors are mediated at the level of mRNA accumulation. The induction of glutamine synthetase mRNA by dibutyryl cyclic AMP was dependent on protein synthesis while the dexamethasone effect was not. Glucocorticoids and cyclic AMP are known to exert their effects on gene expression by different molecular mechanisms. Possible crosstalk between these effector pathways may occur in regulation of astrocyte glutamine synthetase expression.Abbreviations used GS glutamine synthetase - dbcAMP dibutyryl cyclic AMP - MEM minimal essential medium - cyx cycloheximide - GRE glucocorticoid response element - CRE cyclic AMP response element     

Biochemical and genetic characterization of a temperature-sensitive, tryptophanyl-transfer ribonucleic acid synthetase mutant of Bacillus subtilis

A temperature-sensitive, 5-fluorotryptophan (5FT)-resistant mutant of Bacillus subtilis was isolated which forms an altered tryptophanyl transfer ribonucleic acid synthetase [l-tryptophan: sRNA ligase (AMP), EC 6.1.1.2]. The mutant grows well at 30 C but not at 42 C. At the latter temperature, protein and ribonucleic acid (RNA) synthesis are abolished while deoxyribonucleic acid (DNA) synthesis proceeds for a considerable time. Tryptophanyl-transfer RNA (tRNA) synthetase activity is not detectable in the extracts of the mutant grown at 30 C whether this activity is measured by the attachment of l-tryptophan to tRNA or the l-tryptophan-dependent exchange of (32)P-pyrophosphate with adenosine triphosphate. Mixing experiments with extracts from the wild type and the mutant have ruled out the presence of an inhibitor or the absence of an activator as possible causes. Attempts to retrieve enzyme activity in vitro by various means (different conditions for cell disruption, addition of l-tryptophan, and adenosine triphosphate to the extraction buffer containing glycerol) were unsuccessful. The mutation in the locus of the tryptophanyl tRNA synthetase (trpS) was mapped on the bacterial chromosome by transformation and transduction. It is located between argC and metA. All temperature-resistant transformants recover wild-type levels of tryptophanyl tRNA synthetase activity and sensitivity to 5FT. Spontaneous revertants to temperature resistance are 5FT sensitive, but their levels of tryptophanyl tRNA synthetase activity and the thermolability of this enzyme in cell-free extracts varies. These revertants do not support the growth of a presumed nonsense mutant of phase SPO-1. Transduction experiments with phage PBS-1 indicated that reversion must be the result of an event at the site of the original mutation or at a site extremely close to it.