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Translation of Trpg in Bacillus Subtilis Is Regulated by the Trp RNA-Binding Attenuation Protein (TRAP)

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Translation of Trpg in Bacillus Subtilis Is Regulated by the Trp RNA-Binding Attenuation Protein (TRAP) JOURNAL OF BACTERIOLOGY, Aug. 1995, p. 4272–4278 Vol. 177, No. 15 0021-9193/95/$04.0010 Copyright 1995, American Society for Microbiology Translation of trpG in Bacillus subtilis Is Regulated by the trp RNA-Binding Attenuation Protein (TRAP) 1 2 2 1 MIN YANG, ANTOINE DE SAIZIEU, ADOLPHUS P. G. M. VAN LOON, AND PAUL GOLLNICK * Department of Biological Sciences, State University of New York at Buffalo, Buffalo, New York 14260,1 and Section Biotechnology, Vitamin and Fine Chemical Division, F. Hoffmann-La Roche Ltd., CH-4002 Basel, Switzerland2 Received 9 March 1995/Accepted 22 May 1995 trpG of Bacillus subtilis encodes a glutamine amidotransferase subunit that is involved in the synthesis of both folic acid and L-tryptophan. Expression of trpG is negatively regulated by tryptophan even though this gene is located within a folic acid biosynthetic operon. Examination of both transcriptional and translational gene fusions to lacZ involving trpG and direct measurements of trpG mRNA levels and TrpG polypeptide accumulation demonstrated that translation of trpG is regulated by tryptophan whereas transcription is not. These studies also show that this regulation is mediated by the trp RNA-binding attenuation protein. Deletion and point mutations indicated that regulation is dependent on a series of G/UAG trinucleotide repeats surrounding the putative ribosome-binding site for trpG. Our results are consistent with a model in which the tryptophan-activated trp RNA-binding attenuation protein and ribosomes compete for binding to trpG mRNA. 2 2 In Bacillus subtilis, there are seven genes involved in trypto- were done with B. subtilis 1012 (leuA8 metB5 rM mM ) and IBC72 (leuA8 2 2 phan biosynthesis. Six of these are clustered in the trpEDCFBA metB5 rM mM mtrB). The mtrB gene was disrupted in strain IBC72 by ho- mologous integration of a chloramphenicol resistance marker (Fig. 1). DNA operon (9), while the other, trpG, is located within a folate fragments containing the 59 or 39 end of mtrB were generated by PCR with operon containing pab, trpG, pabC, and sul (26). trpG encodes oligonucleotide primers AB1 to AB4 (Table 2). The PCR product of primers a glutamine amidotransferase subunit which is involved in the AB1 and AB2 introduces a TGA stop codon after the first 19 amino acids of mtrB biosynthesis of both folic acid and tryptophan (14). The p- and creates an EcoRI site. The 39 mtrB fragment was created by PCR with aminobenzoate synthase enzyme in the folic acid synthesis primers AB3 and AB4 (Table 2), which also introduced an EcoRI restriction site at the 59 end of the fragment. The two PCR fragments were cloned into pBlue- pathway is composed of subunits of TrpG and Pab, whereas script SK1 (Stratagene), and then a chloramphenicol resistance cassette from anthranilate synthase, which catalyzes the first step in trypto- pBEST401 (12) was inserted into the EcoRI site between the 59 and 39 mtrB phan biosynthesis, is a complex of TrpG and TrpE polypep- fragments to create pFP2A (Fig. 1). This plasmid was linearized with ScaI and tides. transformed into wild-type B. subtilis 1012, and chloramphenicol-resistant trans- Expression of the trp operon is negatively regulated in re- formants were selected. Transformants in which the mtrB gene was disrupted by homologous recombination were identified by Southern blotting. All such mu- sponse to tryptophan by a transcription attenuation mecha- tants were found to result from double-crossover events and did not contain any nism involving the trp RNA-binding attenuation protein vector sequences. The resulting strain was named IBC72. (TRAP) (4, 6, 15, 24), which is the product of the mtrB gene E. coli was transformed by the calcium heat shock procedure (5), and trans- (7). Expression of trpG is also negatively regulated by trypto- formants were selected on Luria-Bertani plates containing either 100 mgof phan (13); however, it does not appear that the other genes in ampicillin per ml or 12.5 mg of chloramphenicol per ml. B. subtilis was trans- formed by using natural competence (1) modified as described in reference 15. the folate operon are controlled by tryptophan. Slock et al. Transformants were selected on plates containing 0.2% acid-hydrolyzed casein, (26) proposed that translation of trpG is regulated in response 0.2% glucose, 10 mgofL-arginine per ml, 13 minimal salts (28), 50 mgof to tryptophan. They suggested that this regulation is mediated 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-Gal) per ml, and 5 mgof by tryptophan-activated TRAP binding to a site in the trpG chloramphenicol per ml. Plasmids and gene fusions. The plasmids used in this study are listed in Table mRNA that overlaps the trpG ribosome-binding site, thus pre- 1. Figure 2 outlines the strategies used to create transcriptional or translational venting ribosomes from initiating translation. In this report, we fusions between trpG and lacZ driven by either the native folate promoter or the demonstrate that while TrpG polypeptide levels vary signifi- heterologous spac promoter (30). cantly in B. subtilis in response to the presence of TRAP, trpG The folate operon promoter has been mapped to approximately 60 bp up- mRNA levels vary only slightly. Furthermore, we used tran- stream of the translational start of pab with the main transcription initiation site at 33 nucleotides upstream of the ATG codon (5a). We used PCR to amplify two scriptional and translational gene fusions between trpG and 1.8-kbp BamHI-BglII fragments containing the promoter, pab, and the first 24 lacZ to demonstrate that tryptophan-activated TRAP does codons of trpG (Fig. 2). The product from oligonucleotides A and G was ligated regulate translation of trpG. into BamHI-digested pDH32 (21) to create a transcriptional fusion with lacZ.In this fusion, a UAA stop codon was created after codon 24 of trpG, which is followed by 61 untranslated nucleotides and then the start codon of lacZ.A MATERIALS AND METHODS translational trpG9-9lacZ fusion was constructed with the PCR product of oligo- nucleotides A and F (Fig. 2). This fragment was ligated into BamHI-cut Bacterial strains and transformations. The bacterial strains used in this study pPDG120, which is similar to pDH32 but contains the polycloning site of pRS552 are described in Table 1. Escherichia coli JM107 was used as the host for plasmid (25) prior to lacZ. constructions, and TG1 was used for site-directed mutagenesis. B. subtilis The spac promoter is a modified version of the E. coli lac promoter that BG2087 (argC4) and BG4233 (argC4 DmtrB) were used as hosts for transforma- functions in B. subtilis (30). We cloned several PCR fragments containing trpG9 tion and integration of gene fusions. BG4233 was created by Dennis Henner under control of this heterologous promoter in pDH88 (8). The trpG9 PCR (Genentech) and contains a deletion of mtrB, which we have determined extends fragments were the products of oligonucleotides C and F (translation fusion) or from codons 7 to 62. Measurements of trpG mRNA and TrpG polypeptide levels C and G (transcriptional fusion) (Fig. 2). After digestion with HindIII-BglII, these fragments contain the first 25 codons of trpG preceded by 71 nucleotides to a naturally occurring HindIII site at position 1668 in pab (26). EcoRI-BglII fragments containing the trpG9 fragments following the spac promoter were * Corresponding author. Phone: (716) 645-2887. Fax: (716) 645- excised from pDH88, and lacZ fusions were constructed as described above for 2975. Electronic mail address: [email protected] fusions under control of the native folate promoter. LO.EDU. We also constructed a translational trpG9-9lacZ fusion to test the effect of 4272 VOL. 177, 1995 TRANSLATION OF trpG IN BACILLUS SUBTILIS 4273 TABLE 1. Bacterial strains and plasmids used in this study. Bacterial strain or Reference Description or genotype plasmid or source E. coli strains JM107 supE44 endA1 hsdR17 gyrA96 relA1 thiD(lac-proAB)29 TG-1 supE hsdD5 thi(lac-proAB), F9(traD36 proAB1 lacIq lacZDM15) B. subtilis strains 2 2 1012 leuA8 metB5 rM mM 22 2 2 r IBC72 leuA8 metB5 rM mM mtrB Cm This study BG2087 argC4 D. Henner BG4233 argC4 DmtrB (deletion from positions 1001 to 1176)a D. Henner r PGBS11 argC4 amyE::[Ppab-(pab-trpG9-9lacZ)] Cm translational fusion This study r PGBS12 argC4 amyE::[Pspac-(9pab-trpG9-9lacZ)] Cm ‘‘coupled’’ translational fusion This study r PGBS13 argC4 amyE::[Pspac-(trpG9-9lacZ)] Cm translational fusion This study r PGBS14 argC4 amyE::[Pspac-(trpG9-9lacZ)] Cm (trpG9236 deletion) This study r PGBS15 argC4 amyE::[Pspac-(trpG9-9lacZ)] Cm (trpG9220 deletion) This study r PGBS16 argC4 amyE::[Pspac-(trpG9-9lacZ)] Cm (trpG9 A-1720 3 G) This study r PGBS17 argC4 amyE::[Pspac-(trpG9-9lacZ)] Cm (trpG9 G-1721 3 T) This study r PGBS18 argC4 amyE::[Pspac-(trpG9-9lacZ)] Cm (trpG9 A-1722 3 G) This study r PGBS19 argC4 amyE::[Ppab-(pab-trpG9-9lacZ)] Cm transcriptional fusion This study r PGBS20 argC4 amyE::[Pspac-lacZ]Cm This study r PGBS21 argC4 amyE::[Pspac-(trpG9-9lacZ)] Cm transcriptional fusion This study r PGBS31 argC4 DmtrB amyE::[Ppab-(pab-trpG9-9lacZ)] Cm translational fusion This study r PGBS32 argC4 DmtrB amyE::[Pspac-(9pab-trpG9-9lacZ)] Cm ‘‘coupled’’ translational fusion This study r PBGS33 argC4 DmtrB amyE::[Pspac-(trpG9-9lacZ)] Cm translational fusion This study r PGBS34 argC4 DmtrB amyE::[Pspac-(trpG9-9lacZ)] Cm (trpG9236 deletion) This study r PBGS35 argC4 DmtrB amyE::[Pspac-(trpG9-9lacZ)] Cm (trpG9220 deletion) This study r PBGS36 argC4 DmtrB amyE::[Pspac-(trpG9-9lacZ)] Cm (trpG9 A-1720 3 G) This
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