The trp RNA-Binding Attenuation Protein Regulates TrpG Synthesis by Binding to the trpg Ribosome Binding Site of Bacillus subtilis

JOURNAL OF BACTERIOLOGY, Apr. 1997, p. 2582 2586 Vol. 179, No. 8 0021-9193/97/$04.00 0 Copyright 1997, American Society for Microbiology The trp RNA-Binding Attenuation Protein Regulates TrpG Synthesis by Binding to the trpg Ribosome Binding Site of Bacillus subtilis HANSEN DU, REX TARPEY, AND PAUL BABITZKE* Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802 Received 23 December 1996/Accepted 10 February 1997 The trpg gene of Bacillus subtilis encodes a glutamine amidotransferase subunit which is involved in the biosynthesis of L-tryptophan and folic acid. The trp RNA-binding attenuation protein (TRAP) is involved in controlling expression of trpg at the level of translation in response to changes in the intracellular concentration of tryptophan. We performed in vitro experiments using purified TRAP to elucidate the mechanism of TRAP-dependent trpg regulation. A TRAP-trpG RNA footprint analysis showed that tryptophan-activated TRAP interacts with one UAG, one AAG, and seven GAG repeats present in the trpg transcript. Results from ribosome and TRAP toeprint experiments indicated that the ribosome and TRAP binding sites overlap. Experiments with a B. subtilis cell-free translation system demonstrated that TRAP inhibits TrpG synthesis. Thus, TRAP regulates translation of trpg by blocking ribosome access to the trpg ribosome binding site. Our results are consistent with a model in which each tryptophan-activated TRAP subunit interacts with one trinucleotide repeat in an RNA target, thereby wrapping the transcript around the periphery of the TRAP complex. Six of the seven genes involved in tryptophan biosynthesis in Bacillus subtilis are linked in the trpedcfba operon (13), whereas trpg is part of a folic acid biosynthetic operon that also includes the pab, pabc, and sul genes (Fig. 1) (21). Expression of the B. subtilis tryptophan biosynthetic genes is regulated by the mtrb-encoded trp RNA-binding attenuation protein (TRAP) (3, 7, 10, 19). The TRAP complex is composed of 11 identical subunits arranged in a single toroid ring (1, 2, 4). TRAP regulates the trp operon by a transcription attenuation mechanism in which tryptophan-activated TRAP binds to 11 closely spaced (G/U)AG repeats present in the nascent trp leader transcript (2, 3, 5, 16, 19, 20). TRAP binding prevents formation of the antiterminator structure, thereby allowing formation of an overlapping rho-independent terminator (see Fig. 2). Thus, TRAP binding promotes transcription termination before RNA polymerase reaches the structural genes of the trp operon. In the absence of TRAP binding, the antiterminator structure prevents formation of the terminator, resulting in transcriptional readthrough into the trp structural genes (see Fig. 2). TRAP also regulates translation of trpe (16, 18). Presumably, TRAP binding to readthrough transcripts promotes refolding of the leader transcript such that the trpe Shine-Dalgarno sequence is sequestered in an RNA hairpin (18). In addition to TRAP controlling expression of the trp operon, recent in vivo experiments demonstrated that TRAP is involved in regulating translation of trpg (24). The TrpG polypeptide functions as the glutamine amidotransferase component of two enzyme complexes. The TrpE-TrpG complex is responsible for formation of o-aminobenzoic acid (anthranilic acid) in the tryptophan biosynthetic pathway, while the Pab- * Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802. Phone: (814) 865-0002. Fax: (814) 863-7024. E-mail: pxb28@psu.edu. TrpG complex is responsible for formation of p-aminobenzoic acid in the biosynthesis of folic acid (12, 14). We performed in vitro experiments to elucidate the mechanism of TRAP-dependent trpg regulation. Our results demonstrate that tryptophanactivated TRAP inhibits TrpG synthesis by binding to nine trinucleotide repeats present in the trpg transcript, thereby blocking ribosome access to the trpg ribosome binding site. MATERIALS AND METHODS Plasmids, bacterial strains, and transformations. Plasmid ppb31 containing trpg was previously described (5). The TRAP-deficient B. subtilis strain, CYBS306, containing a tetracycline resistance gene inserted in the middle of mtrb was described by Merino et al. (18). Transformations and DNA isolation were performed by standard procedures (7). TRAP-trpG RNA footprint. The TRAP-trpG RNA footprint experiments were carried out by modifying a published procedure (5). TRAP was purified as previously described (3). trpg transcripts used in the analysis were synthesized in vitro with the Ambion MEGAscript transcription kit with plasmid ppb31 that had been linearized with ClaI as a template, resulting in a transcript that extended from nucleotides (nt) 72 to 109 relative to the A residue in the trpg AUG start codon (see Fig. 3) (21). Transcripts were gel purified on a 10% denaturing polyacrylamide gel. Reaction mixtures (0.1 ml) contained 2 g (approximately 18 pmol) of TRAP, 1 pmol of unlabeled transcript, 5 g of trna, 10 U of RNasin, 1 mm L-tryptophan, and 1 mm dithiothreitol (DTT) in TKM buffer (5). TRAP-RNA complexes were allowed to form for 10 min at 37 C, at which time 0.5 U of RNase T 1 was added, and the mixtures were incubated for another 10 min at 37 C. Samples were immediately extracted with an equal volume of phenol-chloroform, and the RNA was recovered by two successive ethanol precipitations. RNA pellets were dried and resuspended in primer extension buffer (5). The RNA samples were hybridized to a trpg-specific - 32 P- end-labeled primer (2 pmol) complementary to nt 16 to 36 relative to the A residue in the trpg AUG start codon (see Fig. 3) (21). Primer extension reactions with avian myeloblastosis virus reverse transcriptase (Life Sciences) were performed as previously described (5), and samples were fractionated on a standard 6% sequencing gel. Control sequencing reactions were carried out with plasmid ppb31 and the same end-labeled oligonucleotide. Ribosome and TRAP toeprints. The 30S ribosomal subunit toeprint reactions were performed as described by Hartz et al. (11) with some modifications. The ribosome toeprint assays (10 l mixtures) were carried out by mixing 0.3 pmol of trpg RNA (see above), 0.6 pmol of - 32 P-end-labeled primer complementary to nt 45 to 63 relative to the A residue in the trpg AUG start codon (see Fig. 3) (21), 0.375 mm each deoxynucleotide triphosphate, 12 pmol of either B. subtilis or Escherichia coli 30S ribosomal subunits, 50 pmol of E. coli trna fmet 2582

VOL. 179, 1997 REGULATION OF TrpG SYNTHESIS BY TRAP 2583 FIG. 1. Folate, mtr, and trp operons. trpg is located within the folate operon (21), while the rest of the trp genes are clustered in the trp operon (13). The mtr operon (10) encodes genes involved in folate biosynthesis (mtra) and in controlling expression of the trp genes (mtrb) (7). mtrb encodes TRAP (3, 19), which is composed of 11 identical subunits (1, 2). TRAP is responsible for regulating expression of the trp operon by transcription attenuation (3, 5, 13, 16, 19) and by a translational control mechanism (16, 18). TRAP also controls trpg expression by a translational control mechanism (reference 24 and this paper). p, the position of the promoter for each operon; dashed lines, the presence of downstream open reading frames of unknown function; black box, the trp leader. FIG. 2. Sequence of the B. subtilis trp operon leader transcript showing the mutually exclusive antiterminator and terminator structures. Secondary-structure predictions were performed with the MFOLD program of the Genetics Computer Group sequence analysis software package based on the method described by Zuker and Steigler (25). Boxed nucleotides mark overlapping segments of the competing secondary structures. GAG and UAG repeat sequences are indicated by boldface type. Numbering is from the start of transcription. (Sigma), and 10 U of Moloney murine leukemia virus reverse transcriptase (U.S. Biochemical) in primer extension buffer. TRAP toeprint reaction mixtures contained 0.3 pmol of trpg mrna, 0.6 pmol of labeled primer (see above), 0.375 mm each deoxynucleotide triphosphate, 3 g of TRAP, 1 mm L-tryptophan, 1 mm DTT, and 10 U of Moloney murine leukemia virus reverse transcriptase in TKM buffer. Samples were incubated for 15 min at 37 C, and the reactions were terminated by adding 6 l of standard sequencing stop solution. Control sequencing reactions were carried out with plasmid ppb31 and the same endlabeled oligonucleotide. Cell-free translation of trpg. The trpg transcripts used in the analysis were synthesized in vitro with the Ambion MEGAscript transcription kit with plasmid ppb31 that had been linearized with EcoRI as a template. TRAP-deficient B. subtilis S30 extracts were prepared from strain CYBS306 (18) according to the method described by Chambliss et al. (8), with one modification. Instead of the cell lysate being stirred to shear cellular DNA, RNase-free DNase I (Sigma) was added to the S30 extract (see below). Handling of in vitro translation reactions closely followed the method described by Dick and Matzura (9). Reaction mixtures (50 l) contained 72 mm Tris-HCl (ph 7.5), 72 mm NH 4 Cl, 10 mm magnesium acetate, 0.1 mm EDTA (ph 7.5), 2.4 mm DTT, 2 mm ATP, 0.1 mm GTP, 0.08 mm calcium folinate, 0.2 mm diisopropylfluorophosphate, 20 mm phosphoenolpyruvate, 35 U of pyruvate kinase per ml, 0.1 mm each amino acid except methionine, S30 extract at a protein concentration of 3 mg/ml, 800 U of DNase I per ml, 500 U of RNasin per ml, 2 pmol of trpg transcript, and 10 Ci of [ 35 S]methionine. To reduce endogenous mrna and DNA, the S30 extract was preincubated with RNase-free DNase I (0.8 U/ l) for 15 min at 37 C prior to the addition of the remaining components. The final reaction mixtures were incubated at 37 C for 30 min. All reactions were terminated by the addition of an equal volume of 2 sodium dodecyl sulfate (SDS) sample buffer (100 mm Tris-HCl [ph 6.8], 20% glycerol, 2% SDS, 0.1% bromophenol blue, 2 M 2-mercaptoethanol). Samples (5 l) were heated at 95 C for 3 min and proteins were fractionated on an SDS 15% polyacrylamide gel. Radiolabeled protein bands were quantified with a PhosphorImager (Molecular Dynamics, Inc.) and the ImageQuant software package. RESULTS Identification of the trpg TRAP binding site. Previous studies showed that the TRAP binding site in the trp operon is composed of 11 (G/U)AG repeats, each separated by 2- or 3-nt spacers (Fig. 2) (2, 4, 5), and that the optimal TRAP target consists of single-stranded RNA containing GAG repeats separated by 2 nt (4, 6). Examination of the trpg nucleotide sequence revealed several (G/U)AG repeats that overlap the proposed trpg ribosome binding site (5), although the nucleotide spacing between adjacent repeats is more variable than that in the trp leader transcript (Fig. 3). Furthermore, the trpg RNA segment containing the triplet repeats is not predicted to form any significant secondary structures (25). In addition, Yang et al. (24) demonstrated the general importance of the triplet repeats in controlling trpg expression in vivo. Thus, it seemed likely that TRAP could bind to this RNA segment. A published filter binding assay was used to demonstrate that tryptophan-activated TRAP can bind to an in vitro-synthesized transcript containing the trpg trinucleotide repeats (data not shown) (5, 6). To determine if the TRAP binding site in the trpg message consists of the (G/U)AG repeats, we performed footprinting experiments with purified TRAP and trpg RNA. Since RNase T 1 cleaves following unpaired G residues and every trinucleotide repeat contains at least one G, we used partial RNase T 1 digestion as a probe for analyzing the TRAP-trpG RNA interaction. RNase T 1 cleavage products were identified by primer extension with reverse transcriptase, which results in reverse transcription products 1 nt shorter than the corresponding band in the control sequencing lane (5). The results of the footprint analysis are shown in Fig. 4 and summarized in Fig. 3. The experiments were performed in the presence and absence of bound TRAP. Note that the background banding pattern observed in each experimental lane was also present in the control lane. Presumably, this background pattern is due to low-level nonspecific termination of avian myeloblastosis virus reverse transcriptase at each nucleotide. Every G residue present in the eight (G/U)AG repeats was protected from RNase T 1 cleavage when TRAP was bound to the trpg transcript. We were surprised to find that TRAP also protected the G residue in an AAG repeat. Previous results had demonstrated that TRAP was unable to bind to a synthetic TRAP target consisting of six AAG repeats (4). Apparently, the AAG repeat can interact with TRAP in this particular context. In addition to protecting the G residues in the trinucleotide repeats from RNase T 1 cleavage, three G residues showed enhanced cleavage in the presence of bound TRAP. One of these residues is located downstream of the last GAG repeat. The other two G residues are in spacer segments between repeats,

2584 DU ET AL. J. BACTERIOL. FIG. 3. Nucleotide sequence surrounding the trpg TRAP and ribosome binding sites. The GAG, UAG, and AAG repeats are indicated in boldface type. The previously proposed Shine-Dalgarno sequence (SD) is underlined (21). The amino acid sequence of the TrpG amino terminus is shown. Asterisks, residues that were protected by bound TRAP from RNase T 1 cleavage (two asterisks, high level of protection; one asterisk, low level of protection);, residues that were enhanced for RNase T 1 cleavage; vertical arrows, positions of the TRAP and ribosome toeprints. suggesting that TRAP binding promotes a conformational change in the RNA and that bound TRAP does not protect spacer nucleotides (Fig. 3 and 4). This interpretation is consistent with results from a previous TRAP-trp leader RNA footprint analysis (5). These results demonstrate that TRAP binds to one AAG, one UAG, and seven GAG repeats that are present in a segment of the trpg transcript that overlaps the proposed trpg ribosome binding site (21). Identification of the trpg ribosome binding site. We performed a ribosome toeprint analysis on the trpg transcript using B. subtilis and E. coli 30S ribosomal subunits to identify the ribosome binding site. It is well established that the 16S FIG. 4. Footprint of the TRAP-trpG RNA complex. trpg RNA was treated with RNase T 1 in the presence ( TRAP and L-trp) or absence ( TRAP and/or L-trp) of bound TRAP. Asterisks, bands corresponding to residues that were protected by bound TRAP from RNase T 1 cleavage (two asterisks, high level of protection; one asterisk, low level of protection);, residues that were enhanced for RNase T 1 cleavage. Sequencing lanes to reveal A, C, G, or U residues are shown. The footprint results are summarized in Fig. 3. rrna of the 30S ribosomal subunit base pairs with the mrna Shine-Dalgarno sequence (22) and that an E. coli 30S ribosomal subunit covers 30 to 35 nt with the translation initiation codon positioned near the middle of this RNA segment (15). The presence of a bound ribosome would block primer extension by reverse transcriptase, resulting in a toeprint band at a position corresponding to the 3 boundary of the ribosome (11). We observed identical trna fmet -dependent toeprint bands using either B. subtilis or E. coli 30S ribosomal subunits (Fig. 5). In both cases, the toeprint band was located 15 nt downstream from the first nucleotide of the AUG initiation codon (Fig. 3 and 5), which is in excellent agreement with previously published E. coli ribosome toeprints (11). To the best of our knowledge, this is the first report of a B. subtilis ribosome toeprint. These results indicate that the relative positionings of E. coli and B. subtilis 30S ribosomal subunits on mrna are similar and support the idea that the absence of ribosomal protein S1 from B. subtilis ribosomes is compensated for by a strong interaction between the ribosome binding site on the message and the 16S rrna present in the 30S ribosomal subunit (23). In addition to performing the ribosome toeprint analysis, we determined the position of the TRAP toeprint (Fig. 5). The TRAP toeprint band corresponded to a position 1 nt upstream of the last GAG repeat (Fig. 3 and 5). Presumably, TRAP interaction with the final GAG repeat is relatively weak and can be disrupted by reverse transcriptase. In agreement with this interpretation was the finding that the final GAG repeat was one of the least protected trinucleotide repeats in the RNA footprint (Fig. 3 and 4). These results demonstrate that the ribosome and TRAP binding sites overlap. Regulation of TrpG synthesis. An in vitro system utilizing a TRAP-deficient B. subtilis S30 extract was developed to determine if TRAP binding to trpg mrna inhibits TrpG synthesis. The predicted molecular mass of TrpG is 21.7 kda (21). With the cell-free translation system, a major protein species of approximately 27 kda that was dependent on the addition of in vitro-generated trpg mrna was produced (Fig. 6, lane 1). No translation products were observed without the addition of trpg mrna (data not shown). Migration of the TrpG polypeptide was similar to what was observed previously in a Western blot analysis (24). Thus, it is apparent that TrpG migrates aberrantly in SDS-polyacrylamide gels. When trpg mrna was preincubated with increasing amounts of tryptophan-activated TRAP prior to the addition of the remaining components of the translation system, a corresponding decrease in the level of TrpG synthesis was observed (Fig. 6, lanes 1 to 4). When the preincubation step was omitted, addition of TRAP to the translation system also resulted in a decrease in TrpG synthesis (Fig. 6, lane 5). Note that it was not possible to perform the control experiment in which TRAP is added in the absence of L-tryptophan, since L-tryptophan is required for TrpG synthesis. In conjunction with the footprint and toeprint results described above, the in vitro translation experiments demonstrate that TRAP binding to the trpg message inhibits TrpG synthe-

VOL. 179, 1997 REGULATION OF TrpG SYNTHESIS BY TRAP 2585 FIG. 6. Regulation of TrpG synthesis by TRAP in cell extracts. The B. subtilis S30 extract used in this analysis was produced from the TRAP-deficient strain CYBS306. trpg mrna was preincubated with increasing amounts of tryptophan-activated TRAP prior to the addition of the remaining components of the in vitro translation system (lanes 1 to 4). The preincubation step was omitted for the sample corresponding to lane 5. The position of the TrpG polypeptide is shown, and the relative levels of TrpG synthesis are indicated at the bottom of each lane. The amount of TrpG synthesized in the absence of TRAP (lane 1) was arbitrarily set to 100. FIG. 5. Ribosome and TRAP toeprint analysis of trpg RNA. Ribosome toeprint experiments were carried out with 30S ribosomal subunits from either E. coli (lanes 3 and 4 from the right) or B. subtilis (lanes 1 and 2 from the right), each in the presence ( ) or absence ( )ofe. coli trna fmet. Arrow on the lower right, position of the ribosome toeprint bands; arrow on the upper right, position of the full-length primer extension (PE) product; arrow on the left, position of the TRAP toeprint band ( TRAP). Sequencing lanes to reveal A, C, G, or U residues are shown. The toeprint results are summarized in Fig. 3. sis by blocking ribosome access to the trpg ribosome binding site. DISCUSSION Several previous studies have established that TRAP is responsible for regulating expression of the trpedcfba operon by transcription attenuation (Fig. 2) (3, 7, 16, 18 20). TRAP binding also regulates translation of trpe, presumably by promoting refolding of the trp leader RNA of readthrough transcripts such that the Shine-Dalgarno sequence is sequestered in a secondary structure (16, 18). The one unlinked trp gene, trpg, is located in an operon primarily concerned with folic acid biosynthesis (Fig. 1) (21). Previous in vivo results showed that expression of trpg was regulated at the level of translation in a TRAP-dependent manner, whereas pab expression was not altered in a TRAP-deficient strain (24). Here, we demonstrate that TRAP binding to the trpg transcript blocks ribosome access to the trpg ribosome binding site. Thus, B. subtilis has evolved three distinct regulatory mechanisms by the strategic positioning of two TRAP binding sites, resulting in the coordinate regulation of all seven tryptophan biosynthetic genes in response to changes in the intracellular concentration of tryptophan. The TRAP-trpG RNA footprint analysis identified nine trinucleotide repeats (seven GAG repeats, one UAG repeat, and one AAG repeat) that are involved in TRAP-trpG RNA interaction (Fig. 3 and 4). Results from a previous study demonstrated that TRAP is unable to bind to an RNA target consisting of six AAG repeats (4). However, the AAG repeat in the trpg TRAP binding site is surrounded by appropriately spaced GAG repeats. It is interesting to note that the TRAP binding site in the Bacillus pumilus trp leader also contains one AAG repeat (4, 17, 24). In this case, the AAG repeat is positioned between a UAG repeat and a GAG repeat. Thus, it appears that the ability of an AAG repeat to contribute to TRAP interaction is context dependent. Since evidence now exists that TRAP can interact with GAG, UAG, and AAG repeats (GAG UAG AAG) and that changing the nucleotide in the second or third position in GAG repeats prevents TRAP binding (4), it appears that the A and G residues present in the second and third positions of the triplet repeats are most important for TRAP-RNA recognition (4, 24). Several differences between the trp leader and trpg TRAP binding sites exist. The TRAP binding site in the trp leader consists of 11 repeats (7 GAG and 4 UAG repeats), while the trpg target contains only 9 (7 GAG, 1 UAG, and 1 AAG repeat) (Fig. 3). In addition, each repeat in the trp leader is separated by 2 or 3 nt, with A and U residues being most prevalent, while only 2 of the 10 spacer segments contain a C residue (Fig. 2). In the case of the trpg binding site, 6 of the spacers consist of 2 nt, while 2 other spacer segments contain 5 or 8 nt, respectively (Fig. 3) (4, 24). Furthermore, although A and U residues are the most common nucleotides in the trpg target spacer segments, 5 of the 8 spacers contain a C (Fig. 3). Previous results from experiments with synthetic RNA targets demonstrated that the optimal TRAP binding site is single stranded, consisting of 11 GAG repeats separated by 2 nt (AU or UU) (4, 6). In addition, we previously showed that C residues in the spacers inhibited TRAP binding (6), that TRAP was unable to bind to transcripts in which each repeat was separated by 1, 3, or 4 nt (4), and that transcripts consisting of only AAG repeats did not allow TRAP binding (4). Thus, it is apparent that the trpg TRAP binding site is suboptimal. One reasonable explanation for the suboptimal trpg TRAP target is

2586 DU ET AL. J. BACTERIOL. to allow some TrpG synthesis in the presence of tryptophanactivated TRAP to maintain folic acid biosynthesis (6, 24). ACKNOWLEDGMENTS We are indebted to Elizabeth Rogers and Paul Lovett for the E. coli and B. subtilis 30S ribosomal subunits, Tina Henkin for discussions about in vitro translation, and Robert Simons for discussions about ribosome toeprints. We also thank Paul Gollnick, Charles Yanofsky, and Subramanian Dharmaraj for critical reading of the manuscript. This work was supported by National Institutes of Health grant GM52840. REFERENCES 1. Antson, A. A., A. M. Brzozowski, E. J. Dodson, Z. Dauter, K. S. Wilson, T. Kurecki, J. Otridge, and P. Gollnick. 1994. 11-fold symmetry of the trp RNA-binding attenuation protein (TRAP) from Bacillus subtilis determined by X-ray analysis. J. Mol. Biol. 244:1 5. 2. Antson, A. A., J. Otridge, A. M. Brzozowski, E. J. Dodson, G. G. Dodson, K. S. Wilson, T. M. Smith, M. Yang, T. Kurecki, and P. Gollnick. 1995. 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