Enzymes of the tryptophan operon of Bacillussubtilis

Hoch, Sallie O.; Anagnostopoulos, C.; Crawford, Irving P.

doi:10.1016/0006-291x(69)90700-1

Cited by 67 publications

(48 citation statements)

References 11 publications

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“…These genes are coordinately regulated (13) by a recently revealed, unusual mechanism (22,41), and the complete nucleotide sequence of the operon has been determined (12). The seventh trp gene is not linked to this operon; it participates in both folate and tryptophan syntheses and is located near the pab gene at 10 degrees on the map (36).…”

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An apparent Bacillus subtilis folic acid biosynthetic operon containing pab, an amphibolic trpG gene, a third gene required for synthesis of para-aminobenzoic acid, and the dihydropteroate synthase gene

et al. 1990

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McDonald and Burke (J. Bacteriol. 149:391-394, 1982) previously cloned a sulfanilamide-resistance gene, sul, residing on a 4.9-kb segment of Bacillus subtilis chromosomal DNA, into plasmid pUB110. In this study we determined the nucleotide sequence of the entire 4.9-kb fragment. Genes identified on the fragment include pab, trpG, pabC, sul, one complete unidentified open reading frame, and one incomplete unidentified open reading frame. The first three of these genes, pab, trpG, and pabC, are required for synthesis of p-aminobenzoic acid. The trpG gene encodes an amphibolic glutamine amidotransferase required for synthesis of both p-aminobenzoate and anthranilate, the latter an intermediate in the tryptophan biosynthetic pathway. The pabC gene may encode a B. subtilis analog of enzyme X, an enzyme needed for p-aminobenzoate synthesis in Escherichia coli. The sul gene probably encodes dihydropteroate synthase, the enzyme responsible for formation of 7,8-dihydropteroate, the immediate precursor of folic acid. All six of the cloned genes are arranged in a single operon. Since all four of the identified genes are needed for folate biosynthesis, we refer to this operon as a folic acid operon. Expression of the trpG gene is known to be negatively controlled by tryptophan. We propose that this regulation is at the level of translation. This hypothesis is supported by the finding of an apparent Mtr-binding site which overlaps with the trpG ribosome-binding site.

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An apparent Bacillus subtilis folic acid biosynthetic operon containing pab, an amphibolic trpG gene, a third gene required for synthesis of para-aminobenzoic acid, and the dihydropteroate synthase gene

et al. 1990

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“…N-(5'-Phosphoribosyl)anthranilate isomerase-indole-3-glycerol-phosphate synthase (PRAI-IGPS) from Escherichia coli is a monomeric bifunctional enzyme of Mr 49,500 (1) (4)(5)(6)(7) and by a multienzyme complex in Brevibacteriumflavum (8). The E. coli enzyme is encoded by the trpCF gene of the tryptophan operon.…”

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Three-dimensional structure of the bifunctional enzyme N-(5'-phosphoribosyl)anthranilate isomerase-indole-3-glycerol-phosphate synthase from Escherichia coli.

Priestle¹,

Grütter²,

White³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

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N-(5'-Phosphoribosyl)anthranilate isomerase-indole-3-glycerol-phosphate synthase from Escherichia coli is a monomeric bifunctional enzyme ofMr 49,500 that catalyzes two sequential reactions in the biosynthesis of tryptophan. The three-dimensional structure ofthe enzyme has been determined at 2.8-A resolution by x-ray crystallography. The two catalytic activities reside on distinct functional domains of similar folding, that of an eightfold parallel ,8-barrel with a-helices on the outside connecting the f8-strands. Both active sites were located with an iodinated substrate analogue and found to be in depressions on the surface of the domains created by the outward-curving loops between the carboxyl termini of the ,8-sheet strands and the subsequent a-helices. They do not face each other, making "channeling" of the substrate between active sites virtually impossible. Despite the structural similarity of the two domains, no significant sequence homology was found when topologically equivalent residues were compared. MATERIALS AND METHODS Crystals of PRAI-IGPS were obtained as described (14,15). They belong to space group P41 with unit cell parameters a = b = 104.7 A, c = 68.0 A, and one molecule per asymmetric unit (67% solvent content). Screening of heavy atom derivatives was done by soaking crystals in a buffered solution of the appropriate heavy atom compound in 3 M ammonium sulfate (Table 1). For potentially useful derivatives, a low-resolution diffractometer data set was collected, while oscillation data sets to 2.5-A resolution were collected for the three best derivatives. One derivative was prepared by diffusing an iodinated form of the substrate analogue N-(5'-phosphoribit-1-yl)anthranilate (rCdRP) into the crystals. 5-Iodo-rCdRP was prepared as described for rCdRP (16) except that 5-iodoanthranilate (Fluka) was used in place of anthranilate as starting material and was dissolved in dioxane instead of in 50% aqueous ethanol.The crystals were mounted in thin-walled glass capillaries. Diffractometer data were collected as described elsewhere (17) with the following modifications: after crystal alignment, a single test reflection was used to determine the direction of the c-axis of the crystal; several crystals were required per data set due to strong radiation sensitivity; a maximum intensity loss of about 30% was accepted; and intensities were measured by c-scan over 0.8-1.5°, depending on mosaic spread.Oscillation data were collected and processed as described elsewhere (18) with the following modifications: a cooling device kept the crystal at about 40C; data were collected over 900; two sheets of film per film pack were used; crystal orientation was checked by still photographs at the beginning and end of data collection; and the films were digitized, using a 3.0 OD scale.The oscillation and diffractometer data sets (Table 1) were not merged at this point. Heavy atom sites were accepted only if found with both sets of data. They were located by difference Patterson and difference Fourier techniqu...

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“…Following the promoter region lies an additional regulatory element, the attenuator, that contains a site for regulated transcriptional termination (5). The tryptophan metabolism of Bacillus subtilis has been extensively studied (6) and the genes for the enzymes of the tryptophan pathway, trpEDCFBA, have been localized to a single area of the genetic map (7). However, for B. subtilis, or other Gram-positive bacteria, no transcriptional regulation system using repressor-operator interaction or attenuator-transcription termination control analogous to that found in E. coli has been described.…”

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Characterization of the Bacillus subtilis tryptophan promoter region.

Shimotsu¹,

Henner²

1984

Proc. Natl. Acad. Sci. U.S.A.

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The nucleotide sequence of the control region of the trp operon of Bacillus subtilis has been determined. The region was shown to contain the tip promoter by deletion analysis and by determination of the transcription start site.The tip promoter shows similarity to the consensus sequence for Escherichia coli and B. subtilis promoters. The presence of the trp control region on a high-copy-number plasmid confers resistance to the tryptophan analogue 5-methyltryptophan. It appears that an approximately 120-base-pair region comprising not only the tip promoter but also adjacent direct repeat sequences is necessary to confer 5-methyltryptophan resistance. We postulate that this region is involved in tryptophan regulation and confers 5-methyltryptophan resistance by titration of a tip regulatory protein. Removal of either the trp promoter or the adjacent direct repeat sequences abolished the 5-methyltryptophan-resistance phenotype. Placement of unrelated promoters adjacent to the direct repeat sequences restored 5-methyltryptophan resistance. This suggests that promoter activity is necessary for the regulatory function.The tryptophan (trp) operon of Escherichia coli has been a model system to investigate operon structure, gene function, and regulation of gene expression (1, 2). From such studies a detailed understanding of the regulation of the trp operon has emerged. The regulatory region of the trp operon consists of a promoter region that contains an operator that binds a tryptophan-activated repressor and regulates transcription (3, 4). Following the promoter region lies an additional regulatory element, the attenuator, that contains a site for regulated transcriptional termination (5). The tryptophan metabolism of Bacillus subtilis has been extensively studied (6) and the genes for the enzymes of the tryptophan pathway, trpEDCFBA, have been localized to a single area of the genetic map (7). However, for B. subtilis, or other Gram-positive bacteria, no transcriptional regulation system using repressor-operator interaction or attenuator-transcription termination control analogous to that found in E. coli has been described. Because B. subtilis has been the most studied Gram-positive organism, we felt that it would be of interest to determine whether such a transcriptional regulation system exists in the trp operon of B. subtilis and to compare its mechanisms of gene regulation with the corresponding mechanisms of E. coli. Recently, we have cloned several overlapping portions of the trp operon of B. subtilis into plasmid pBR322. The nucleotide sequence of the entire trp operon has been determined (ref. 8; unpublished data). In this paper, we report the nucleotide sequence of the control region of the trp operon of B. subtilis. The promoter was identified and the transcriptional start site was determined. In addition, we have identified a region that, when placed on a high-copy-number plasmid, confers resistance to the tryptophan analogue 5-methyltryptophan (5-MeTrp). We postulate that this region is involved...

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Enzymes of the tryptophan operon of Bacillussubtilis

Cited by 67 publications

References 11 publications

An apparent Bacillus subtilis folic acid biosynthetic operon containing pab, an amphibolic trpG gene, a third gene required for synthesis of para-aminobenzoic acid, and the dihydropteroate synthase gene

An apparent Bacillus subtilis folic acid biosynthetic operon containing pab, an amphibolic trpG gene, a third gene required for synthesis of para-aminobenzoic acid, and the dihydropteroate synthase gene

Three-dimensional structure of the bifunctional enzyme N-(5'-phosphoribosyl)anthranilate isomerase-indole-3-glycerol-phosphate synthase from Escherichia coli.

Characterization of the Bacillus subtilis tryptophan promoter region.

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