The complete nucleotide sequence of the tryptophan operon of Escherichia coli

Yanofsky, Charles; Platt, Terry; Crawford, Irving P.; Nichols, Brian P.; Christie, Gail E.; Horowitz, Heidi; vanCleemput, Magda; Wu, Anna M.

doi:10.1093/nar/9.24.6647

Cited by 336 publications

(146 citation statements)

References 59 publications

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“…In general, similar codon preferences were observed among these reading frames, with some notable exceptions: for example, UUA and CUG for Leu were used most frequently in all but tdcC, where CUG was preferred; AUC for Ile was the dominant codon in tdcC; AAU was the most frequently used codon for Asn in tdcA and ORFX, whereas AAC was preferred in tdcB and tdcC; and GGU for Gly was used largely in tdcA. A comparison of average codon preference values for the five genes of the E. coli trp operon (27) with that of the three tdc reading frames and of ORFX shows some differences in the average frequency of codon usage (Table 3). A calculation of codon preference statistics, P (which is a measure of bias in codon usage), with the bacterial highly expressed codon usage data of Grantham et al (8), yielded P values of 0.870, 0.967, and 0.973 for tdcA, tdcB, and tdcC, respectively.…”

Section: Methodsmentioning

confidence: 99%

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Molecular characterization of the tdc operon of Escherichia coli K-12

1988

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The nucleotide sequence of a 2-kilobase DNA fragment of the tdc region of Escherichia coli K-12, previously cloned in this laboratory, revealed two open reading frames, tdcC and ORFX, downstream from the tdcB gene (formerly designated tdc) encoding biodegradative threonine dehydratase. A 24-base-pair sequence separated tdcC from the dehydratase coding region, and an untranslated region of 60 nucleotides, which contains a recognizable -10 consensus sequence, was found between tdcC and ORFX. The deduced amino acid sequence of tdcC showed it to be a large hydrophobic polypeptide of 431 amino acid residues, whereas ORFX coded for a small 135-residue polypeptide lacking glutamine and tryptophan. A computer-assisted sequence analysis revealed no similarity among the tdcB, tdcC, and ORFX polypeptides, and a search of the GenBank database failed to detect similarity with any other known proteins. The tdc genes and ORFX showed similar codon usage and, in analogy with other bacterial genes, showed codon usage typical for genes expressed at an intermediate level. Transcriptional analysis with S1 nuclease indicated two distinct transcription start sites upstream of the tdcB gene in regions previously identified as promoterlike elements P1 and P2. Interestingly, expression of tdcB and tdcC, but not ORFX, was contingent upon the presence of P1. These results taken together tend to suggest that the biodegradative threonine dehydratase is the second gene in a polycistronic transcription unit constituting a novel operon (tdcABC) in E. coli implicated in anaerobic threonine metabolism.Biodegradative threonine dehydratase (EC 4.2.1.16) of Escherichia coli catalyzes the pyridoxal phosphate-dependent dehydration of L-threonine and L-serine to ammonia and to a-ketobutyrate and pyruvate, respectively (22). The enzyme is normally induced under anaerobic culture conditions in tryptone-yeast extract medium lacking fermentable carbohydrates. Recently, Hobert and Datta (11) devised a synthetic medium consisting of four amino acids, threonine, serine, valine, and isoleucine, plus cyclic AMP and fumarate, which supported higher levels of dehydratase synthesis as compared with that found in the rich medium with or without cyclic AMP and fumarate. Because the requirements for cyclic AMP and fumarate were absolute in terms of enzyme synthesis, it was proposed that not the amino acids themselves but some metabolite(s) derived from these amino acids during anaerobic metabolism was needed for dehydratase induction. In addition, Merberg and Datta (17) reported that various strains of E. coli with unrelated genotypes produced various levels of the enzyme in the same medium and that mutations at multiple loci on the chromosome influenced enzyme expression in vivo. These cumulative findings led to the notion that a complex network of regulatory systems controls the synthesis of dehydratase in anaerobic culture conditions.To understand the mechanism of dehydratase induction at the DNA level, we cloned a 6.2-kilobase-pair (kb) DNA fragment of E. coli ...

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Section: Methodsmentioning

confidence: 99%

“…Thus, the polypeptides coded by tdcA, tdcC, and ORFX represent three new proteins in E. coli whose catalytic characteristics and metabolic roles remain to be uncovered. (10) 42 (14) 22 (2) 42 Ser UCU 23 (7) 26 (6) 29 (11) 12 (2) 15 UCC 16 (5) 35 (8) 29 (11) (27) are given.…”

Section: Methodsmentioning

confidence: 99%

Molecular characterization of the tdc operon of Escherichia coli K-12

1988

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“…Reaction (2) is catalyzed by the fl2 subunit and 50-fold more efficiently by the complex. This mutual activation is thought to arise from heterologous subunit-subunit interactions during assembly of the complex.…”

Section: -Phosphatementioning

confidence: 99%

“…Meanwhile the nucleotide sequences of the corresponding genes have been determined for E. roli [2], other enteric bacteria [3], and for Saccharomyces cerevisiae [4], showing that the amino acid sequences of the CI and / 3 polypeptide chains are highly conserved. Further, limited proteolysis has revealed that both the (x subunit ( M , 28727) and the p protomer (M, 42988) are composed of two autonomously folding structural domains that probably form the corresponding active sites at the interdomain interfaces [5 -91.…”

Section: -Phosphatementioning

confidence: 99%

Small‐Angle X‐Ray Scattering Studies of Tryptophan Synthase from Escherichia coli and Its α and β₂ Subunits

Wilhelm

Pilz

Lane

et al. 1982

European Journal of Biochemistry

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The a and 0 2 subunits of tryptophan synthase were investigated by small-angle X-ray scattering. The molecular parameters are: radius of gyration, CI: 1.95 nm, pz : 3.01 nm; maximum particle diameter, CI: 5.8 nm, p 2 : 10.5 nm; and hydrated volume, CI : 60 nm3, p2 160 nm'. The shape of the CI subunit can best be described by a circular cylinder, slightly tapered at one end. An elongated elliptical cylinder with its cross section larger in the middle than at the ends was found to be a model equivalent in scattering to the p2 subunit.The CI& enzyme complex was found to have a radius of gyration of 4.01 nm, a maximum length of 13.5 nm, and a hydrated volume of 270 nm3. No satisfactory fit of the scattering data was obtainable by mere apposition of the models of the CI and p 2 subunits. Two cylinders overlapping laterally fit the experimental data considerably better, suggesting changes in the conformation of the subunits on forming the CI& complex.Tryptophan synthase from Escherichia coli is an CI& bienzyme complex, which catalyses the following reactions :Reaction (1) is catalyzed by the CI subunit and 100-fold more efficiently by the (x& complex. Reaction (2) is catalyzed by the fl2 subunit and 50-fold more efficiently by the complex. This mutual activation is thought to arise from heterologous subunit-subunit interactions during assembly of the complex. Moreover, the active sites appear to be juxtaposed at the intersubunit interface [l].The information on the structure and function of the complex and the component subunits has been reviewed recently [I]. Meanwhile the nucleotide sequences of the corresponding genes have been determined for E. roli [2], other enteric bacteria [3], and for Saccharomyces cerevisiae [4], showing that the amino acid sequences of the CI and / 3 polypeptide chains are highly conserved. Further, limited proteolysis has revealed that both the (x subunit ( M , 28727) and the p protomer (M, 42988) are composed of two autonomously folding structural domains that probably form the corresponding active sites at the interdomain interfaces [5 -91. Except for a preliminary crystallographic study of the a subunit [lo] and an estimate of the distance between the two active sites [Ill, little direct evidence on the tertiary and quaternary structure of tryptophan synthase and its subunits is available to date.In this paper we present the results of small-angle X-ray scattering studies on the CI and p2 subunits and the a& complex of tryptophan synthase. The data were fitted to various models equivalent in scattering and support a scheme for the assembly of the complex from its subunits. MATERIALS AND METHODS EnzymesTryptophan synthase (x-& complex was purified from the overproducing strain Escherichia coli trpR trpAEDl02/ F'trpAEDl02 [12]. The excess CI subunit produced by this strain was purified by affinity chromatography [13]. The p 2 subunit was prepared from the pure CI& complex by heat denaturation as described [12,14]. Protein aggregates were removed by gel chromatography on Sephacryl S200 (...

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“…The Nterminus of the R. graveo/ensAS(xl precursor was arbitrarily chosen as the first amino acid of the numbering scheme. Sources of sequences are: Arabidopsis thaliana, Niyogi and Fink (1992); Saccharomyces cerevisiae, Zalkin et al (1984), corrected as suggested by Crawford (1999) from positions 615 to 629; Salmonella typhimurium, Caliguri and Bauerle (1991); Escherichia coIL Yanofsky et aL (1981). The conserved sequence motif L(I./F)ES (131-134) is critical for allosteric inhibition by tryptophan (Matsui et al, 1987).…”

Section: Molecular Mass Of Native Asmentioning

confidence: 99%

Purification and cDNA cloning of anthranilate synthase from Ruta graveolens: modes of expression and properties of native and recombinant enzymes

et al. 1995

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SummaryRuta graveolens utilizes anthrenilete synthese (AS) for the synthesis both of tryptophan in primary metabolism and acridone alkaloids in secondary metabolism. AS has been purified from plants and cell cultures of R. graveolens 670-and 1700-fold, respectively. Glutamine-end ammoniadependent AS activities were strictly co-purified in all steps. Through cDNA cloning and complementetion of Escherichia coli deletion mutants defective for AS, it is shown that young Ruta plants express two genes for functional AS~ subunits, AS~ land AS(~2. The data indicate that AS~ from Ruta requires an AS~ subunit with a native molecular weight of 60-65 kDe for the glutaminedependent reaction. Protein synthesized in vitro from cloned cDNA is processed upon import into isolated chloroplasts, indicating that mature AS~ subunits are active in plastids in vivo. AS~I and AS(~2 are constitutively expressed in Ruta cell cultures, but AS~I steedy-stete mRNA levels are increased 100-fold 6 h subsequent to elicitation whereas AS~2 expression remains constitutive. Increased AS~I transcription corresponds to elicitorinduced alkaloid accumulation. The data indicate that Ruta regulates anthranilate flux into primary and secondary metabolism through differential regulation of AS genes specific to these pathways.

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The complete nucleotide sequence of the tryptophan operon of Escherichia coli

Cited by 336 publications

References 59 publications

Molecular characterization of the tdc operon of Escherichia coli K-12

Molecular characterization of the tdc operon of Escherichia coli K-12

Small‐Angle X‐Ray Scattering Studies of Tryptophan Synthase from Escherichia coli and Its α and β₂ Subunits

Purification and cDNA cloning of anthranilate synthase from Ruta graveolens: modes of expression and properties of native and recombinant enzymes

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The complete nucleotide sequence of the tryptophan operon of Escherichia coli

Cited by 336 publications

References 59 publications

Molecular characterization of the tdc operon of Escherichia coli K-12

Molecular characterization of the tdc operon of Escherichia coli K-12

Small‐Angle X‐Ray Scattering Studies of Tryptophan Synthase from Escherichia coli and Its α and β2 Subunits

Purification and cDNA cloning of anthranilate synthase from Ruta graveolens: modes of expression and properties of native and recombinant enzymes

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Small‐Angle X‐Ray Scattering Studies of Tryptophan Synthase from Escherichia coli and Its α and β₂ Subunits