Temperature-induced derepression of tryptophan biosynthesis in a tryptophanyl-transfer ribonucleic acid synthetase mutant of Bacillus subtilis

A tryptophanyl-transfer ribonucleic acid (tRNA) synthetase (l-tryptophan: tRNA ligase adenosine monophosphate, EC 6.1.1.2) mutant (trpS1) of Bacillus subtilis is derepressed for enzymes of the tryptophan biosynthetic pathway at temperatures which reduce the growth rate but still allow exponential growth. Derepression of anthranilate synthase in a tryptophan-supplemented medium (50 mug/ml) is maximal at 36 C, and the differential rate of synthesis is 600- to 2,000-fold greater than that of the wild-type strain or trpS1 revertants. A study of the derepression pattern in the mutant and its revertants indicates that the 5-fluorotryptophan recognition site of the tryptophanyl-tRNA synthetase is an integral part of the repression mechanism. Evidence for a second locus, unlinked to the trpS1 locus, which functions in the repression of tryptophan biosynthetic enzymes is presented.     

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.     

Selection of cell lines resistant to tryptophan analogs

After prolonged cultivation in the presence of increasing amounts of carboxyl-substituted tryptophan analogs (tryptamine and tryptophanol), cell lines resistant to high concentrations of these compounds were obtained. The initial culture was the Madin-Darby line of spontaneously transformed bovine kidney cells. In the resistant lines the amount of tryptophanyl-tRNA synthetase (E. C. 6.1.1.2) is manyfold increased as shown by two criteria: (i) enzymatic activity (ATP-PPi isotopic exchange) per mg of protein, (ii) binding of in vivo 35S-labeled proteins to polyclonal antibodies against tryptophanyl-tRNA synthetase. It was shown that tryptophanyl-tRNA synthetase is phosphorylated in vivo, and the degree of phosphorylation of the enzyme in initial cells seems to be higher then in the resistant ones. The Km value for tryptophan is not significantly changed for the enzyme from resistant cells. The permeability for tryptophan and its analogs is reduced in the resistant cells. It is proposed that the acquisition of the resistance against tryptophan analogs are due to alterations at the genomic level (for example, gene amplification etc.).     

Mischarging mutants of Su+2 glutamine tRNA in E. coli. II. Amino acid specificities of the mutant tRNAs

Among the mischarging mutants isolated from strains with Su+2 glutamine tRNA, two double-mutants, A37A29 and A37C38, have been suggested to insert tryptophan at the UAG amber mutation site as determined by the suppression patterns of a set of tester mutants of bacteria and phages (Yamao et al., 1988). In this paper, we screened temperature sensitive mutants of E. coli in which the mischarging suppression was abolished even at the permissive temperature. Four such mutants were obtained and they were identified as the mutants of a structural gene for tryptophanyl-tRNA synthetase (trpS). Authentic trpS mutations, such as trpS5 or trpS18, also restricted the mischarging suppression. These results strongly support the previous prediction that the mutant tRNAs of Su+2, A37A29 and A37C38, are capable of interacting with tryptophanyl-tRNA synthetase and being misaminoacylated with tryptophan in vivo. However, in an assay to determine the specificity of the mutant glutamin tRNAs, we detected predominantly glutamine, but not any other amino acid, being inserted at an amber codon in vivo to any significant degree. We conclude that the mutant tRNAs still accept mostly glutamine, but can accept tryptophan in an extent for mischarging suppression. Since the amber suppressors of Su+7 tryptophan tRNA and the mischarging mutants of Su+3 tyrosine tRNA are charged with glutamine, structural similarity among the tRNAs for glutamine, tryptophan and tyrosine is discussed.     

Human tryptophanyl-tRNA synthetase is switched to a tRNA-dependent mode for tryptophan activation by mutations at V85 and I311

For most aminoacyl-tRNA synthetases (aaRS), their cognate tRNA is not obligatory to catalyze amino acid activation, with the exception of four class I (aaRS): arginyl-tRNA synthetase, glutamyl-tRNA synthetase, glutaminyl-tRNA synthetase and class I lysyl-tRNA synthetase. Furthermore, for arginyl-, glutamyl- and glutaminyl-tRNA synthetase, the integrated 3' end of the tRNA is necessary to activate the ATP-PPi exchange reaction. Tryptophanyl-tRNA synthetase is a class I aaRS that catalyzes tryptophan activation in the absence of its cognate tRNA. Here we describe mutations located at the appended beta1-beta2 hairpin and the AIDQ sequence of human tryptophanyl-tRNA synthetase that switch this enzyme to a tRNA-dependent mode in the tryptophan activation step. For some mutant enzymes, ATP-PPi exchange activity was completely lacking in the absence of tRNA(Trp), which could be partially rescued by adding tRNA(Trp), even if it had been oxidized by sodium periodate. Therefore, these mutant enzymes have strong similarity to arginyl-tRNA synthetase, glutaminyl-tRNA synthetase and glutamyl-tRNA synthetase in their mode of amino acid activation. The results suggest that an aaRS that does not normally require tRNA for amino acid activation can be switched to a tRNA-dependent mode.     

Tryptophanyl-tRNA synthetase from Bacillus subtilis. Characterization and role of hydrophobicity in substrate recognition

The tryptophanyl-tRNA synthetase from Bacillus subtilis was purified to homogeneity and characterized. It has an alpha 2 subunit structure and a molecular weight of 77,000. Tryptophanyl-tRNA synthetase does not catalyze any significant proofreading. It activates tryptophan as well as the three fluorinated analogues, DL-4-fluoro-, DL-5-fluoro-, or DL-6-fluorotryptophan (4F-, 5F-, and 6F-Trp), in the ATP-pyrophosphate exchange reaction. In the aminoacylation reaction, the fluorotryptophans act as competitive inhibitors of Trp. Their relative activities follow the same order in both reactions: Trp greater than 4F-Trp greater than 6F-Trp greater than 5F-Trp. This order is the inverse of the order of relative hydrophobicities of these compounds, pointing to the importance of hydrophobic interactions in the selective recognition by tryptophanyl-tRNA synthetase among this group of substrates. To define the physical basis of the relative hydrophobicities, the crystallographic structure of 4F-Trp was determined and compared to that of trptophan. Charge distributions calculated for tryptophan and its different fluoroanalogues on the basis of molecular structures were supported by their carbon-13 NMR spectra. Correlations between charge distributions and relative hydrophobicities suggest that the polarity of the C-F bond represents an underlying factor determining the hydrophobicities of 4F-, 5F-, and 6F-Trp, thus relating tryptophanyl-tRNA synthetase selectivity toward tryptophan and its fluoroanalogues directly to their electronic configurations.     

Temperature-sensitive repression of the tryptophan operon in Escherichia coli

Mutants of Escherichia coli exhibiting temperature-sensitive repression of the tryptophan operon have been isolated among the revertants of a tryptophan auxotroph, trpS5, that produces an altered tryptophanyl transfer ribonucleic acid (tRNA) synthetase. Unlike the parental strain, these mutants grew in the absence of tryptophan at high but not at low temperature. When grown at 43.5 C with excess tryptophan (repression conditions), they produced 10 times more anthranilate synthetase than when grown at 36 C or lower temperatures. Similar, though less striking, temperature-sensitivity was observed with respect to the formation of tryptophan synthetase. Transduction mapping by phage P1 revealed that these mutants carry a mutation cotransducible with thr at 60 to 80%, in addition to trpS5, and that the former mutation is primarily responsible for the temperature-sensitive repression. These results suggest that the present mutants represent a novel type of mutation of the classical regulatory gene trpR, which probably determines the structure of a protein involved in repression of the tryptophan operon. In agreement with this conclusion, tRNA of several trpR mutants was found to be normal with respect to its tryptophan acceptability. It was also shown that the trpS5 allele, whether present in trpR or trpR(+) strains, produced appreciably higher amounts of anthranilate synthetase than the corresponding trpS(+) strains under repression conditions. This was particularly true at higher temperatures. These results provide further evidence for our previous conclusion that tryptophanyl-tRNA synthetase is somehow involved in repression of this operon.     

Evidence for Compartmentation of Tryptophan in Cultured Plant Tissues: Free Tryptophan Levels and Inhibition of Anthranilate Synthetase

1. Anthranilate synthetase activity in crude extracts from tissue cultures of Daucus carota L. (carrot), Nicotiana tabacum L. (tobacco; cv. Wisconsin 38 and xanthi), Glycine max Merr. (soybean) and Oryza sativa L. (rice) was completely inhibited by l -tryptophan (5 to 50 μM). Mutant carrot and tobacco lines, capable of growth in the presence of 5-methyltryptophan, required 500 to more than 1000 μM tryptophan for complete inhibition of enzyme activity, respectively. 2. Except for the mutant tobacco line, the concentrations of free tryptophan in all tissue cultures tested were greater than the levels necessary to completely inhibit the respective anthranilate synthetase activities in vitro. These findings would indicate that much of the free tryptophan is compartmentalized away from the regulatory enzyme, anthranilate synthetase. This could implicate compartmentalization of the inhibitor as a biosynthetic control mechanism. 3. During the growth of normal and mutant carrot tissues the anthranilate synthetase enzyme must be at least 7.8 and 10.8% active, respectively, in order to accumulate the amount of tryptophan found in the tissues. 4. Of the substrates and cofactors required for anthranilate synthetase activity in vitro, Mg²⁺ and glutamine were present at near optimal levels in the carrot and tobacco tissues, but chorismate was found to be significantly below the optimal concentrations.     

Intrachromosomal gene mapping in man: the gene for tryptophyl-tRNA synthetase maps in region q21 leads to qter of chromosome 14

A gene for tryptophanyl-tRNA synthetase (EC 6.1.1.2), the enzyme which attaches tryptophan to its tRNA, has previously been assigned to human chromosome 14 by analysis of man-mouse somatic cell hybrids. We report here a method for the electrophoretic separation of Chinese hamster and human tryptophanyl-tRNA synthetases and its application to a series of independently derived Chinese hamster-human hybrids in which part of the human chromosome 14 has been translocated to the human X chromosome. When this derivative der (X),t(X;14) (Xqter leads to Xp22::14q21 leads to 14qter) chromosome carrying the human gene for hypoxanthine-guanine phosphoribosyltransferase was selected for and against in cell hybrid lines by the appropriate selective conditions, the human tryptophanyl-tRNA synthetase activity was found to segregate concordantly. These results provide additional confirmation for the assignment of the tryptophanyl-tRNA synthetase gene to human chromosome 14 and define its intrachromosomal location in the region 14q21 leads to 14qter. Our findings indicate that the genes for tryptophanyl-tRNA synthetase and for ribosomal RNA are not closely linked on chromosome 14.     

Mutational identification of an essential tryptophan in tryptophanyl-tRNA synthetase of Bacillus subtilis.

The strongly conserved single tryptophan residue, Trp92, in Bacillus subtilis tryptophanyl-tRNA synthetase has been mutagenized via site direction singly into Gln, Ala, and Phe. All three mutant enzymes were inactive toward the catalysis of tRNA tryptophanylation. The Trp92----Phe mutant has been subcloned into the high expression plasmid pKK223-3 to yield the recombinant plasmid pKSW-F92. Growth of bacteria carrying the latter plasmid made possible the purification of the mutant TrpRS-F92 enzyme to homogeneity. This mutant enzyme was deficient in ultraviolet absorbance and fluorescence relative to the wild type enzyme and inactive in the partial reaction of Trp-activation as well as the overall reaction of tRNA tryptophanylation. Furthermore, unlike the wild type B. subtilis trpS gene, the mutant trpS-F92 gene upon transformation into Escherichia coli trpS 10343 failed to complement the temperature sensitive trpS mutation of the host cells. Trp92 therefore represents an essential residue both in vitro and in vivo for the function of the tryptophanyl-tRNA synthetase.     

Tryptophanyl-tRNA synthetase is found closely associated with and stimulates DNA polymerase α-like activity from wheat embryos

DNA polymerase α-like from wheat embryos is found to purify closely associated with a tryptophanyl-tRNA synthetase activity. No other aminoacyl-tRNA synthetases were present. A purified preparation of wheat tryptophanyl-tRNA synthetase free of polymerase activity was able to stimulate plant DNA polymerase of the α-like type, while the γ-like polymerase from wheat embryos was not affected by the enzyme. We have not been able to find a diadenosine 5′, 5′′′-P¹,P⁴-tetraphosphate binding activity associated to the polymerase-synthetase complex. We have also observed a specific inhibition by beef tRNA^Trp of DNA polymerase α-like activity, while other tRNAs will not change the enzyme activity.     

Regulation of Tryptophan Biosynthetic Enzymes in Neurospora crassa

The formation of enzymatic activities involved in the biosynthesis of tryptophan in Neurospora crassa was examined under various conditions in several strains. With growth-limiting tryptophan, the formation of four enzymatic activities, anthranilic acid synthetase (AAS), anthranilate-5-phosphoribosylpyrophosphate phosphoribosyl transferase (PRAT), indoleglycerol phosphate synthetase (InGPS), and tryptophan synthetase (TS) did not occur coordinately. AAS and TS activities began to increase immediately, whereas PRAT and InGPS activities began to increase only after 6 to 12 hr of incubation. In the presence of amitrole (3-amino-1,2,4-triazole), the formation of TS activity in a wild-type strain was more greatly enhanced than were AAS and InGPS activities. With a tr-3 mutant, which ordinarily exhibits an elevated TS activity, amitrole did not produce an increase in TS activity greater than that observed on limiting tryptophan. With tr-3 mutants, the increased levels of TS activity could be correlated with the accumulation of indoleglycerol in the medium; prior genetic blocks which prevented or reduced the synthesis of indoleglycerol also reduced the formation of TS activity. The addition of indoleglycerol to cultures of a double mutant (tr-1, tr-3) which could not synthesize indoleglycerol markedly stimulated the production of TS activity but not PRAT activity; the production of TS activity reached the same level with limiting or with excess tryptophan. A model explaining these and other related observations on enzyme formation in N. crassa is proposed.     

Selection and characterization of Escherichia coli variants capable of growth on an otherwise toxic tryptophan analogue

Escherichia coli isolates that were tolerant of incorporation of high proportions of 4-fluorotryptophan were evolved by serial growth. The resultant strain still preferred tryptophan for growth but showed improved growth relative to the parental strain on other tryptophan analogues. Evolved clones fully substituted fluorotryptophan for tryptophan in their proteomes within the limits of mass spectral and amino acid analyses. Of the genes sequenced, many genes were found to be unaltered in the evolved strain; however, three genes encoding enzymes involved in tryptophan uptake and utilization were altered: the aromatic amino acid permease (aroP) and tryptophanyl-tRNA synthetase (trpS) contained several amino acid substitutions, and the tyrosine repressor (tyrR) had a nonsense mutation. While kinetic analysis of the tryptophanyl-tRNA synthetase suggests discrimination against 4-fluorotryptophan, an analysis of the incorporation and growth patterns of the evolved bacteria suggest that other mutations also aid in the adaptation to the tryptophan analogue. These results suggest that the incorporation of unnatural amino acids into organismal proteomes may be possible but that extensive evolution may be required to reoptimize proteins and metabolism to accommodate such analogues.     

Substrate depletion analysis as an approach to the pre-steady-state anticooperative kinetics of aminoacyl adenylate formation by tryptophanyl-tRNA synthetase from beef pancreas

The formation of tryptophanyl adenylate catalyzed by tryptophanyl-tRNA synthetase from beef pancreas has been studied by stopped-flow analysis under conditions where the concentration of one of the substrates was largely decreasing during the time course of the reaction. Under such conditions a nonlinear regression analysis of the formation of the adenylate (adenylate vs. time curve) at several initial tryptophan and enzyme concentrations gave an accurate determination of both binding constants of this substrate. The use of the jackknife procedure according to Cornish - Bowden & Wong [ Cornish - Bowden , A., & Wong , J.J. (1978) Biochem. J. 175, 969-976] gave the limit of confidence of these constants. This approach confirmed that tryptophanyl-tRNA synthetase presents a kinetic anticooperativity toward tryptophan in the activation reaction that closely parallels the anticooperativity found for tryptophan binding at equilibrium. Both sites are simultaneously forming the adenylate. The dissociation constants obtained under the present pre-steady-state conditions for tryptophan are KT1 = 1.6 +/- 0.5 microM and KT2 = 18.5 +/- 3.0 microM at pH 8.0, 25 degrees C. The rate constant kf of adenylate formation is identical for both active sites (kf = 42 +/- 5 s-1). The substrate depletion method presently used, linked to the jackknife procedure, proves to be particularly suitable for the determination of the kinetic constants and for the discrimination between different possible kinetic models of dimeric enzyme with high substrate affinity. In such a case this method is more reliable than the conventional method using substrate concentrations in high excess over that of the enzyme.     

Assignment of a gene for tryptophanyl-transfer ribonucleic acid synthetase (E.C. 6.1.1.2) to human chromosome 14

We describe a simple method for locating tryptophanyl-tRNA synthetase (E.C. 6.1.1.2) on cellulose acetate gels (Cellogel) following electrophoresis. Employing electrophoretic conditions which result in the separation of mouse and human tryptophanyl-tRNA synthetases, we have analyzed extracts of a number of independently derived mouse-human somatic cell hybrids and subclones derived from these hybrids for the presence of human tryptophanyl-tRNA synthetase. Electrophoretic patterns of hybrid extracts which contain human tryptophanyl-tRNA synthetase exhibit three bands. This is consistent with published evidence that the enzyme from mammalian cells is a homologous dimer. The electrophoretic patterns derived from some hybrids are unusual in that the human and hybrid bands of activity are more intense than the mouse band from the same hybrid. An analysis of hybrid cells and extracts indicates that human tryptophanyl-tRNA synthetase segregates with human chromosome 14 and with the only enzyme marker which has previously been assigned to this chromosome, nucleoside phosphorylase.R. M. D. was supported by a postdoctoral fellowship from the Damon Runyon Fund for Cancer Research. The work described was supported in part by grants from Cancer Research Campaign, the Medical Research Council, and NATO.     

Isolation of the novel regulatory mutants of the tryptophan biosynthetic system in Escherichia coli

Summary A novel type of tryptophan requiring mutants of Escherichia coli was isolated. The mutation maps between str and malA.These mutants, designated as trpS, have alterations in the regulation of the tryptophan operon. Neither derepression nor complete repression of the tryptophan biosynthetic enzymes was observed with this mutant. Dominance test shows that the trpS mutation is recessive to the wild type allele. TrpS mutant, therefore, is a type of super-repressed mutants distinct from i ^s mutant in the lactose system of E. coli.It was found that the tryptophanyl-tRNA synthetase is specified by the trpS gene. This indicates that the transfer mechanism of tryptophan is related to repression of the tryptophan operon.     

Altered prephenate dehydratase in phenylalanine-excreting mutants of Brevibacterium flavum.

The regulatory properties of three key enzymes in the phenylalanine biosynthetic pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase (DAHP synthetase) [EC 4.1.2.15], chorismate mutase [EC 5.4.99.5], and prephenate dehydratase [prephenate hydro-lyase (decarboxylating), EC 4.2.1.51] were compared in three phenylalanine-excreting mutants and the wild strain of Brevibacterium flavum. Regulation of DAHP synthetase by phenylalanine and tyrosine in these mutants did not change at all, but the specific activities of the mutant cell extracts increased 1.3- to 2.8-fold, as reported previously (1). Chorismate mutase activities in both the wild and the mutant strains were cumulatively inhibited by phenylalanine and tyrosine and recovered with tryptophan, while the specific activities of the mutants increased 1.3- to 2.8-fold, like those of DAHP synthetase. On the other hand, the specific activities of prephenate dehydratase in the mutant and wild strains were similar, when tyrosine was present. While prephenate dehydratase of the wild strain was inhibited by phenylalanine, tryptophan, and several phenylalanine analogues, the mutant enzymes were not inhibited at all but were activated by these effectors. Tyrosine activated the mutant enzymes much more strongly than the wild-type enzyme: in mutant 221-43, 1 mM tyrosine caused 28-fold activation. Km and the activation constant for tyrosine were slightly altered to a half and 6-fold compared with the wild-type enzyme, respectively, while the activation constants for phenylalanine and tryptophan were 500-fold higher than the respective inhibition constants of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2 x 10(5), a half of that of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2 X 10(5) a half of that of the wild type enzyme, while in the presence of tyrosine, phenylalanine, or tryptophan, it increased to that of the wild-type enzyme. Immediately after the mutant enzyme had been activated by tyrosine and then the tyrosine removed, it still showed about 10-fold higher specific activity than before the activation by tyrosine. However, on standing in ice the activity gradually fell to the initial level before the activation by tyrosine. Ammonium sulfate promoted the decrease of the activity. On the basis of these results, regulatory mechanisms for phenylalanine biosynthesis in vivo as well as mechanisms for the phenylalanine overproduction in the mutants are discussed.     

Genetic control of tryptophan hypersynthesis in regulatory mutants of Pseudomonas putida

The 6-fluorotryptophan resistant MR1 mutant was obtained from Pseudomonas putida M30 (Tyr- Phe-) strain. The mutant was able to excrete tryptophan (60 micrograms/ml) and has derepressed aroF gene encoding 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase. The mutation isolated was identified as aroR with the help of cloning early aroF gene of P. putida. On the next step of selection, regulatory mutant MR2 was obtained producing 240 micrograms/ml of tryptophan. The MR2 has derepressed unlinked trpE and trpDC genes and represents a mutant of the trpR type. Expression of the trpE gene of P. putida MR2 weakened in the presence of tryptophan excess in the medium, which points to attenuation of this gene. From the prototrophic variant of P. putida MR2 the MRP3 mutant producing 850 micrograms/ml of tryptophan was obtained. This mutant was characterized by twofold increase in the activity of the anthranilate synthase encoded by the trpE gene. The assay of the activity of tryptophanyl-tRNA synthase in P. putida MRP3 demonstrated that the mutant has TrpS+ phenotype.