JOURNAL OF BACTERIOLOGY, Jan. 2004, p. 278–286 Vol. 186, No. 20021-9193/04/$08.00�0 DOI: 10.1128/JB.186.2.278–286.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

The trp RNA-Binding Attenuation Protein of Bacillus subtilisRegulates Translation of the Tryptophan Transport Gene

trpP (yhaG) by Blocking Ribosome BindingHelen Yakhnin,† Hong Zhang,† Alexander V. Yakhnin, and Paul Babitzke*

Department of Biochemistry and Molecular Biology, The Pennsylvania State University,University Park, Pennsylvania 16802

Received 9 September 2003/Accepted 22 October 2003

Expression of the Bacillus subtilis tryptophan biosynthetic genes (trpEDCFBA and pabA [trpG]) is regulatedin response to tryptophan by TRAP, the trp RNA-binding attenuation protein. TRAP-mediated regulation of thetryptophan biosynthetic genes includes a transcription attenuation and two distinct translation control mech-anisms. TRAP also regulates translation of trpP (yhaG), a single-gene operon that encodes a putative trypto-phan transporter. Its translation initiation region contains triplet repeats typical of TRAP-regulated mRNAs.We found that regulation of trpP and pabA is unaltered in a rho mutant strain. Results from filter binding andgel mobility shift assays demonstrated that TRAP binds specifically to a segment of the trpP transcript thatincludes the untranslated leader and translation initiation region. While the affinities of TRAP for the trpP andpabA transcripts are similar, TRAP-mediated translation control of trpP is much more extensive than for pabA.RNA footprinting revealed that the trpP TRAP binding site consists of nine triplet repeats (five GAG, threeUAG, and one AAG) that surround and overlap the trpP Shine-Dalgarno (S-D) sequence and translation startcodon. Results from toeprint and RNA-directed cell-free translation experiments indicated that tryptophan-activated TRAP inhibits TrpP synthesis by preventing binding of a 30S ribosomal subunit. Taken together, ourresults establish that TRAP regulates translation of trpP by blocking ribosome binding. Thus, TRAP coordi-nately regulates tryptophan synthesis and transport by three distinct mechanisms: attenuation transcriptionof the trpEDCFBA operon, promoting formation of the trpE S-D blocking hairpin, and blocking ribosomebinding to the pabA and trpP transcripts.

The Bacillus subtilis trpEDCFBA operon contains six of theseven genes required for the biosynthesis of tryptophan fromchorismic acid, the common aromatic amino acid precursor(reviewed in references 4 and 17). pabA (trpG), the remainingtryptophan biosynthetic gene, is present in an operon primarilyconcerned with folic acid biosynthesis (31). Expression of thetrp operon and pabA is regulated in response to tryptophan bythe trp RNA-binding attenuation protein (TRAP) (4, 17).TRAP is composed of 11 identical subunits arranged in a singlering (2). TRAP-mediated regulation of the trp operon includestranscription attenuation and translation control mechanisms.The trp operon leader transcript contains inverted repeats thatallow folding of the transcript to form several RNA secondarystructures that participate in the attenuation mechanism. Twoof these structures, the antiterminator and terminator, overlapby 4 nucleotides (nt) and therefore are mutually exclusive (3, 6,9, 22, 26). Tryptophan-activated TRAP can bind to 11 tripletrepeats (7 GAG and 4 UAG) present in the nascent trp leadertranscript (Fig. 1) (7). Because six of these repeats are presentwithin the RNA segment that folds into the antiterminatorstructure, TRAP binding prevents formation of this structureby wrapping the RNA around the outside of the protein ring(1). As a consequence, formation of the overlapping termina-

tor is favored, which causes transcription termination beforeRNA polymerase can reach the first gene in the operon (trpE).In the absence of TRAP binding, formation of the antitermi-nator allows transcription of the entire operon. Since TRAPmust bind before RNA polymerase transcribes past the termi-nator, the timing of TRAP binding is crucial for this regulatorydecision. NusA-stimulated RNA polymerase pausing providesadditional time for TRAP to bind to the nascent trp operontranscript, thereby increasing the termination efficiency at theattenuator (38).

In addition to regulating trp operon expression by transcrip-tion attenuation, TRAP regulates translation of trpE. TRAPbinding to trp operon readthrough transcripts promotes forma-tion of an RNA hairpin that sequesters the trpE Shine-Dal-garno (S-D) sequence, thereby reducing TrpE synthesis byinhibiting ribosome binding (14, 22, 24). Formation of the trpES-D blocking hairpin also reduces expression of the secondgene in the operon (trpD) via translational coupling and tran-scriptional polarity (40). A Mg2�-dependent RNA tertiarystructure that forms in the trp operon readthrough transcript iscapable of sequestering all 11 (G/U)AG repeats. This tertiarystructure appears to interfere with TRAP-mediated translationcontrol of trpE by inhibiting TRAP binding to preexistingreadthrough transcripts (29).

pabA is the second gene in an operon primarily concernedwith folic acid biosynthesis. The PabA polypeptide functions asa glutamine amidotransferase in the biosynthesis of tryptophanand folic acid (21). Translation of pabA is regulated by TRAPin response to tryptophan (41). TRAP binds to nine triplet

* Corresponding author. Mailing address: Department of Biochem-istry and Molecular Biology, The Pennsylvania State University, Uni-versity Park, PA 16802. Phone: (814) 865-0002. Fax: (814) 863-7024.E-mail: pxb28@psu.edu.

† H.Y. and H.Z. contributed equally to this work.

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repeats (seven GAG, one UAG, and one AAG) that surroundand overlap the pabA S-D sequence. Bound TRAP inhibitsPabA synthesis by blocking ribosome access to the pabA ribo-some binding site (Fig. 1) (16).

In addition to the tryptophan biosynthetic genes, TRAPregulates expression of yhaG, a gene that encodes an apparenttryptophan transporter (27). Because of its likely involvementin tryptophan transport, we propose to rename the gene trpP toreflect this function. The trpP transcript contains nine tripletrepeats (five GAG, three UAG, and one AAG) that surroundand overlap its S-D sequence and translation initiation region(Fig. 1). Previous in vivo expression studies demonstrated thatTRAP regulates translation of trpP. We performed experi-ments in vitro to elucidate the mechanism responsible forTRAP-dependent regulation of trpP. Our results establish thatTRAP regulates TrpP synthesis by blocking ribosome binding.

MATERIALS AND METHODS

Bacterial strains and plasmids. The plasmids pPB77 containing the B. subtilistrp operon leader (3), pPB31 containing pabA (7), pYH14 containing a rho::neoallele (40), and pYH28 containing a trpE�-�gfp translational fusion in whichtryptophan codon 57 in gfp was changed to a phenylalanine codon (29) have beendescribed previously. The cloning vectors pTZ19R and pTZ18U each contain aT7 RNA polymerase promoter upstream from a polylinker (United States Bio-chemical Corp.). The plasmid pHZB6 was constructed by cloning a chromo-somally derived PCR fragment containing �1 to �137 relative to the start of trpPtranscription into pTZ18U. Plasmid pYH34 was constructed by replacing the trpoperon sequences in pYH28 with a chromosomally derived PCR fragment con-taining �1 to �112 relative to the start of trpP transcription. The resultingtrpP�-�gfp translational fusion contained the 10th trpP codon fused in frame withthe first gfp codon.

The B. subtilis strains used in this study are described in Table 1. B. subtilisstrain PLBS338 was constructed by transforming strain 168 (trpC2) with W168(tryptophan prototroph) chromosomal DNA, selecting for tryptophan prototro-phy, and screening for rifampin sensitivity (0.25 �g/ml). Strains PLBS339 andPLBS340 were constructed by transforming PLBS338 with chromosomalDNA from CYBS400::pJS648 (amyE::PtrpPtrpP�-�lacZ Cmr) and PGBS11(amyE::PpabpabB-pabA�-�lacZ Cmr), respectively. Selection was for chloram-phenicol resistance (5 �g/ml). Integration into amyE was confirmed by screeningfor the absence of amylase activity (30). Transforming strains PLBS339 andPLBS340 with chromosomal DNA from strain BG4233 (�mtrB) resulted instrains PLBS341 and PLBS342, respectively. Selection was for 5-fluorotrypto-phan resistance (200 �g/ml). rho::neo from pYH14 was used to replace thewild-type rho allele in strains PLBS339, PLBS340, PLBS341, and PLBS342 toyield strains PLBS343, PLBS344, PLBS345, and PLBS346, respectively. Selectionwas for kanamycin resistance (10 �g/ml).

�-Galactosidase assays. B. subtilis cultures were grown in minimal-acid caseinhydrolysate medium containing 5 �g of chloramphenicol/ml in the absence orpresence of 200 �M tryptophan. Growth medium for rho mutant strains alsocontained 10 �g of kanamycin/ml. The cells were harvested during late expo-nential phase. Aliquots were then assayed for �-galactosidase activity as previ-ously described (14).

Gel mobility shift assay. TRAP was purified as previously described (39).Quantitative gel mobility shift assays used to examine TRAP-RNA interactionsfollowed a previously published procedure (39). RNA was synthesized in vitrousing the Ambion MEGAscript kit. Linearized plasmid pPB31 was used as thetemplate to generate pabA RNA containing nt �72 to �109 relative to the AUGstart codon. Linearized plasmid pHZB6 was used to synthesize trpP RNA con-taining nt �82 to �55 relative to the AUG start codon (�1 to �137 relative tothe start of transcription). Gel-purified transcripts were dephosphorylated andsubsequently 5�-end labeled with T4 polynucleotide kinase and [�-32P]ATP.Labeled transcripts were gel purified, ethanol precipitated, and suspended inTris-EDTA (TE). Transcripts were renatured by heating to 80°C for 1 minfollowed by slow cooling prior to use in binding reactions.

FIG. 1. Comparison of the known TRAP binding sites. The triplet repeats are shown in bold type. The S-D sequences and the translation startcodons (Met) are shown for pabA, trpP, and ycbK. The pabB and rtpA stop codons overlap the S-D sequences of pabA and ycbK, respectively. ycbKis a gene of unknown function (28). The trp operon, pabA, trpP, and ycbK sequences shown in this figure correspond to nt �36 to �91, �1405 to�1456, �36 to �101, and �519 to �578 relative to the start of transcription, respectively.

TABLE 1. B. subtilis strains used in this study

Strain Genotypea Source

168 trpC2 BGSCb

BG4233 �mtrB argC4 20CYBS400::pJS648 amyE::PtrpPtrpP�-�lacZ Cmr 27CYBS306 mtrB�Tc amyE::PtrptrpE�-�lacZ Cmr 24PGBS11 argC4 amyE::PpabpabB-pabA�-�lacZ Cmr 41PLBS338 Prototroph This studyPLBS339 amyE::PtrpPtrpP�-�lacZ Cmr This studyPLBS340 amyE::PpabpabB-pabA�-�lacZ Cmr This studyPLBS341 �mtrB amyE::PtrpPtrpP�-�lacZ Cmr This studyPLBS342 �mtrB amyE::PpabpabB-pabA�-�lacZ Cmr This studyPLBS343 rho::neo (Kmr) amyE::PtrpPtrpP�-�lacZ Cmr This studyPLBS344 rho::neo (Kmr) amyE::PpabpabB-pabA�-

�lacZ CmrThis study

PLBS345 �mtrB rho::neo amyE::PtrpPtrpP�-�lacZ Cmr This studyPLBS346 �mtrB rho::neo amyE::PpabpabB-pabA�-

�lacZ CmrThis study

W168 rpoB18 (Rifr) BGSC

a Ptrp, Ppab, and PtrpP denote the trpEDCFBA, folate, and trpP operon promot-ers, respectively. A prime indicates truncation of the gene.

b BGSC, Bacillus subtilis Genetic Stock Center, Ohio State University, Colum-bus, Ohio.

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Binding reaction mixtures (8 �l) contained 50 mM Tris-acetate (pH 8.0), 4mM magnesium acetate, 5 mM dithiothreitol (DTT), 10% glycerol, 0.2 mg ofEscherichia coli tRNA/ml, 400 U of RNasin (Promega)/ml, 0.1 nM 5�-end-labeled pabA RNA or 0.5 nM 5�-end-labeled trpP RNA, 1.2 mM L-tryptophan,purified TRAP (various concentrations), and 0.1 mg of xylene cyanol/ml. Com-petition assays also contained unlabeled RNA competitor (see Results for de-tails). TRAP-RNA complexes were allowed to equilibrate at 37°C for 20 min.Samples were then fractionated on native 6% (pabA) or 8% (trpP) polyacryl-amide gels in 375 mM Tris-HCl (pH 8.8), 5% glycerol, and 1 mM EDTA.Radioactive bands were visualized using a PhosphorImager (Molecular Dynam-ics). Free and bound RNA species were quantified using ImageQuant (Molec-ular Dynamics), and the apparent equilibrium binding constants (Kd) of TRAP-RNA complexes were calculated by fitting to the simple binding equation aspreviously described (39).

Filter binding assay. The labeled transcripts used in filter binding reactionswere identical to those described for the gel mobility shift assay. Filter bindingassays were carried out using a 96-well dot blot apparatus by modifying a two-filter method reported previously (36). Filters were equilibrated in 40 mM Tris-HCl (pH 8.0) and 250 mM KCl prior to use. Binding reaction mixtures (45 �l)contained 40 mM Tris-HCl (pH 8.0), 250 mM KCl, 5 mM DTT, 0.2 mg of E. colitRNA/ml, 400 U of RNasin/ml, 0.1 nM 5�-end-labeled RNA, 1.2 mM L-trypto-phan, and purified TRAP (various concentrations). TRAP-RNA complexes wereallowed to equilibrate at 37°C for 20 min. Samples (40 �l) were then filtered andsubsequently rinsed twice with 100 �l of 40 mM Tris-HCl (pH 8.0) and 250 mMKCl. Radioactive spots were visualized and quantified as described for the gelmobility shift assay.

Footprint assay. 5�-End-labeled trpP RNA used in this analysis was generatedas described for the gel mobility shift assay. Titrations of RNase T1 (Roche),RNase T2 (Sigma), RNase A (Ambion), and RNase V1 (Pierce) were performedto optimize the amount of each reagent to prevent multiple cleavages in any onetranscript. RNA suspended in TE was renatured by heating to 80°C for 1 minfollowed by slow cooling. Binding reaction mixtures (10 �l) contained 40 mMTris-HCl (pH 8.0), 30 mM KCl, 8 mM MgCl2, 32.5 ng of total yeast RNA, 100�g of bovine serum albumin/ml, 7.5% glycerol, 1 mM L-tryptophan, 2 nM trpPRNA, and various concentrations of TRAP. Reaction mixtures were incubatedfor 30 min at 37°C to allow TRAP-trpP RNA complex formation prior to theaddition of RNase T1 (8 10�3 U/�l), RNase T2 (2 10�4 U/�l), RNase V1 (3 10�5 U/�l), or RNase A (10�6 �g/�l). Incubation was then continued for 15min at 37°C. Reactions were terminated by the addition of 5 �l of stop solution(95% formamide, 20 mM EDTA, 0.025% sodium dodecyl sulfate [SDS], 0.025%xylene cyanol, 0.025% bromophenol blue), and samples were fractionatedthrough 6% sequencing gels. Radiolabeled bands were visualized by phospho-rimagery.

Toeprint assay. Toeprint assays were performed by modifying published pro-cedures (10, 14, 19). trpP RNA was synthesized using linearized pHZB6 astemplate. Gel-purified RNA (250 nM) in TE was hybridized to a 32P-end-labeledDNA oligonucleotide (500 nM) complementary to the 3� end of the transcript byheating to 80°C for 1 min and slow cooling. Toeprint assays were carried out with3 �M TRAP and 1 mM L-tryptophan and/or 10 pmol of E. coli 30S ribosomalsubunits and 50 pmol of E. coli tRNAfMet (Sigma). Toeprint reaction mixtures(10 �l) contained 2 �l of the hybridization mixture, a 375 �M concentration ofeach deoxynucleoside triphosphate, 10 mM DTT, and 100 �g of bovine serumalbumin/ml in toeprint buffer (50 mM Tris-HCl [pH 8.3], 75 mM KCl, 3 mMMgCl2). TRAP toeprint reactions were incubated for 30 min at 37°C to allowTRAP-trpP RNA complex formation. 30S ribosomal subunit toeprint reactions

were performed by incubating RNA with 30S ribosomal subunits and tRNAfMet

as described previously (19). After the addition of 10 U of Moloney murineleukemia virus reverse transcriptase (U.S. Biochemical), incubation was contin-ued at 37°C for 15 min. Reactions were terminated by the addition of 6 �l of stopsolution (see “Footprint assay”). Samples were fractionated through 6% se-quencing gels. Sequencing reactions were performed using pHZB6 as the tem-plate and the same end-labeled DNA oligonucleotide as a primer. Radiolabeledbands were visualized by phosphorimagery.

RNA-directed cell-free translation. The trpP�-�gfp translational fusion tran-script used for this analysis was synthesized using linearized pYH34 as template.A TRAP-deficient B. subtilis S-30 extract was prepared from strain CYBS306 byfollowing a published procedure (12). Cell-free translation reactions were carriedout by modifying published procedures (14, 16, 29). The S-30 extract was prein-cubated with RNase-free DNase I for 15 min at 37°C to remove endogenousmRNA and DNA. Reaction mixtures (24 �l) contained 60 mM Tris-HEPES (pH7.5), 60 mM NH4Cl, 15 mM MgCl2, 12 mM KCl, 0.5 mM EGTA, 5 mM DTT, 2mM ATP, 0.6 mM GTP, 0.08 mM calcium folinate, 4 �g of aprotinin/ml, 4 �g ofleupeptin/ml, 4 �g of pepstatin A/ml, 4 �l of S-30 extract (12 �g of total protein),800 U of DNase I/ml, 500 U of RNasin/ml, 10 mM phosphoenolpyruvate, 35 Uof pyruvate kinase/ml, 0.4 mg of E. coli tRNA/ml, 100 nM trpP�-�gfp mRNA, 10�Ci of [35S]methionine, 5 mM potassium glutamate, 5 mM glutamine, and a 0.1mM concentration of each of the other amino acids except tryptophan. Trypto-phan was added at a concentration of 1 mM when used. Reaction mixtures wereincubated for 25 min at 37°C and terminated by adding 6 �l of SDS-stop buffer(125 mM Tris-HCl [pH 6.8], 5% SDS, 25% glycerol, 2% 2-mercaptoethanol, and12.5 mg of bromophenol blue/ml). Aliquots (10 �l) were heated at 95°C for 5min, and proteins were fractionated on SDS–14% polyacrylamide gels. Radio-labeled bands were visualized by phosphorimagery and quantified using Image-Quant.

RESULTSTRAP-mediated regulation of trpP and pabA expression.

Previous results demonstrated that TRAP regulates translationof trpP (27) and pabA (16, 41). We compared TRAP-mediatedregulation of these two genes by measuring �-galactosidaseactivities in strains containing pabA�-�lacZ or trpP�-�lacZ trans-lational fusions that were otherwise isogenic. The effect ofexogenous tryptophan was assessed from the ratio of expres-sion when cells were grown in the absence or presence oftryptophan (�Trp/�Trp ratio). TRAP-dependent regulationwas observed for both fusions with inhibition ratios (�Trp/�Trp) of 5 for pabA and 150 for trpP (Table 2). As expected,when these experiments were repeated in a TRAP-deficientgenetic background (�mtrB), regulation in response to trypto-phan was abolished (Table 2). The extent of TRAP-mediatedregulation in vivo was determined by comparing expression in�mtrB strains with that in wild-type strains grown in the pres-ence of tryptophan. The TRAP-dependent inhibition ratios forpabA and trpP were 16 (545/34) and 900 (1,200/1.3), respec-tively (Table 2). These results indicate that TRAP-mediated

TABLE 2. Regulation of trpP and pabA expression

Fusion Relevantgenotype

�-Galactosidase activityaInhibition ratio(�Trp/�Trp) mtrB/WTb

�Trp �Trp

pabA�-�lacZ Wild type 178 10 34 4 5.2 16pabA�-�lacZ mtrB 545 55 518 38 1.1pabA�-�lacZ rho 99 10 16 3 6.2 27pabA�-�lacZ mtrB rho 432 22 450 13 1.0trpP�-�lacZ Wild type 201 33 1.3 0.3 155 923trpP�-�lacZ mtrB 1,200 160 1,200 220 1.0trpP�-�lacZ rho 184 39 1.4 0.4 131 929trpP�-�lacZ mtrB rho 1,300 230 1,340 170 1.0

a �-Galactosidase activity is given in Miller units (25).b The extent of TRAP-dependent inhibition was determined by dividing the value for the mtrB mutant by the value for the corresponding wild-type strain (WT) �Trp.

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regulation of trpP expression is more tightly controlled thanthat of pabA.

TRAP binding to trp operon readthrough transcripts regu-lates TrpE synthesis by promoting formation of an RNA sec-ondary structure that sequesters the trpE S-D sequence (14). Itwas also shown that Rho causes transcriptional polarity of thetrp operon under conditions that promote translation control(tryptophan excess); expression was elevated in rho mutantstrains (40). Since both trpP and pabA are regulated by TRAPat the level of translation, we carried out experiments to de-termine whether expression of these two genes was altered ina rho mutant background. Expression from the trpP�-�lacZfusion was unchanged in the Rho-deficient strain, whereasexpression from the pabA�-�lacZ fusion was reduced twofold inthe rho mutant strain (Table 2). When expression from eachfusion was examined in mtrB rho double mutant strains, �-ga-lactosidase activity was similar to that observed in the mtrBsingle mutant strains. These results indicate that transcrip-tional polarity, if present, is not sufficient to provide regulation.

TRAP binds to trpP and pabA RNA with comparable affin-ities. The mechanism of TRAP-mediated inhibition of PabAsynthesis was previously characterized; however, the affinity ofTRAP for the pabA transcript was not examined. To charac-terize further the interaction of tryptophan-activated TRAPwith pabA mRNA, we performed quantitative gel mobility shiftassays with a pabA transcript containing nt �72 to �109 rela-tive to its AUG start codon (Fig. 2A). Nonlinear least-squaresanalysis of these data yielded an estimated equilibrium bindingconstant (Kd) of 14 nM TRAP. Filter binding studies were alsocarried out as an alternative method to measure the affinity oftryptophan-activated TRAP for pabA RNA. This methodyielded a Kd value of 33 nM TRAP. For comparison, the Kd

value for the TRAP-trp leader RNA interaction was found tobe approximately 7 nM by gel mobility shift analysis (data notshown) and 1 nM by filter binding (42). Similar gel mobilityshift and filter binding assays were also carried out to examineTRAP interaction with a trpP transcript containing nt �82 to�55 relative to the AUG start codon (�1 to �137 relative tothe start of transcription). In this case, estimated Kd values of51 and 31 nM TRAP were obtained for gel mobility shift (Fig.2C) and filter binding, respectively. It is important to note thatwhile the affinities of TRAP for pabA and trpP RNA weresimilar, the extent of TRAP-mediated regulation of the twogenes differed considerably (Table 2). Possible explanations forthis apparent discrepancy will be addressed in the Discussion.

While the above binding studies determined the affinity ofTRAP for the pabA and trpP transcripts, the specificities of theTRAP-pabA RNA and TRAP-trpP RNA interactions were in-vestigated by performing competition experiments with spe-cific and nonspecific unlabeled RNA competitors. The concen-tration of TRAP used in these experiments was chosen suchthat about 75% of the labeled RNA was shifted in the absenceof competitor RNA (Fig. 2B, second lane, and D, second lane).As expected, pabA RNA was an effective competitor forTRAP-pabA RNA interaction, whereas a transcript derivedfrom pTZ19R vector sequences was not (Fig. 2B). The speci-ficity of TRAP-trpP RNA interaction was investigated by per-forming competition experiments with specific (trpP, trpL, andpabA) and nonspecific unlabeled competitors (Fig. 2D). Thetrp leader (trpL) transcript was the most effective competitor,

while competition levels with the pabA and trpP transcriptswere comparable to one another. As expected, the nonspecificcompetitor (pTZ19R) was unable to compete for TRAP-trpPRNA complex formation. These results establish that TRAPbinds specifically to a trpP transcript containing its S-D se-quence and translation initiation region.

TRAP binds to nine triplet repeats in the trpP transcript.Previous footprinting studies demonstrated that TRAP bindsto 11 repeats in the trp operon leader transcript (7 GAG and 4UAG) and to 9 repeats in the pabA message (7 GAG, 1 UAG,and 1 AAG). In general, the spacer residues separating adja-cent repeats were not protected by bound TRAP (7, 16). Otherpublished results have indicated that the affinity of TRAP ishighest for GAG repeats (GAG � UAG � AAG � CAG) and

FIG. 2. Gel mobility shift analysis of TRAP complexed with pabAand trpP transcripts. 5�-End-labeled RNA was incubated with the con-centration of TRAP shown at the bottom of each lane. Gel shift assayswere performed in the absence or presence of various competitorRNAs. The concentration of each competitor RNA is indicated at thebottom of the corresponding lane. The positions of bound and freeRNA are shown. (A) TRAP-pabA RNA complex formation. (B) Com-petition assay for TRAP-pabA RNA complex formation. (C) TRAP-trpP RNA complex formation. (D) Competition assay for TRAP-trpPRNA complex formation.

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that optimal spacing between repeats was 2 nt, with pyrimi-dines generally favored over purines (5, 8, 11, 37).

It was previously pointed out that nine triplet repeats werepresent in the trpP transcript that overlapped and surroundedthe trpP S-D sequence and translation initiation region (fiveGAG, three UAG, and one AAG) (Fig. 1) (27). However, inseveral cases the length of the spacers was suboptimal, rangingin size from 1 to 14 nt. Footprint experiments were carried outto determine which of the nine repeats constituted authenticTRAP recognition targets. We used partial RNase T1 (cleavesfollowing single-stranded G residues), RNase T2 (preferen-tially cleaves following single-stranded A residues), and RNaseA (cleaves following single-stranded pyrimidines) digestion toprobe the TRAP-trpP RNA complex. The use of these threereagents would theoretically be capable of cleaving every nu-cleotide in the trpP transcript. The results of the footprintanalysis are shown in Fig. 3 and are summarized in Fig. 4.RNase T1 cleaved the majority of the G residues in the ninerepeats in the absence of bound TRAP. Notable exceptionswere the Gs in repeats 2 and 7. Tryptophan-activated (bound)TRAP protected G residues in repeats 1, 3, 4, 6, and 8 fromRNase T1 cleavage, whereas bound TRAP caused enhancedcleavage of the G residue in repeat 9. RNase T2 cleaved the

central A residue in repeats 1, 3, 5, 6, 8, and 9 in the absenceof bound TRAP. Importantly, TRAP protected all of these Aresidues from RNase T2 cleavage. RNase A cleaved the Uresidues in repeats 4, 7, and 9 in the absence of bound TRAP,while bound TRAP prevented cleavage at these three nucleo-tides. Thus, with the exception of repeat 2, the use of thesethree reagents allowed us to identify TRAP-dependent protec-tion of each repeat. In addition to protecting residues withinthe triplet repeats, bound TRAP protected 7 out of 39 spacernucleotides. In contrast, bound TRAP resulted in enhancedcleavage of seven spacer residues (Fig. 3 and 4). Thus, as waspreviously observed for the TRAP binding targets in the trpoperon leader and in pabA, TRAP generally protected theresidues in the triplet repeats but not those in the spacersseparating the repeats.

The RNA segment between nt 40 and 55 was refractory toRNase cleavage, suggesting that an RNA structure was presentin the transcript. Computer predictions using MFOLD version3.1 (23, 43) identified a large secondary structure extendingfrom G18 through C54. Interestingly, the first triplet repeat ispresent in the loop of this hairpin and was extensively cleavedby RNases T1 and T2. Since the second trinucleotide repeat ispresent in the 3� half of the stem within the secondary struc-

FIG. 3. TRAP-trpP RNA footprint analysis. trpP RNA was treated with RNase T1, RNase T2, or RNase A in the absence or presence of TRAP.The concentrations of TRAP used were 0, 0.25, 0.5, 1, and 2 �M. Partial alkaline hydrolysis (OH) and RNase T1 digestion (T1) ladders, as wellas control (C) lanes in the absence of RNase treatment, are shown. The RNase T1 ladder was generated under denaturing conditions so that everyG residue in the transcript could be visualized. Residues in which RNase cleavage was reduced (�) or enhanced (�) in the presence of TRAPare marked. Apparent TRAP-dependent RNase T1 cleavage of C90 is marked with an arrowhead. The relative positions of the triplet repeats (1to 9), as well as the S-D sequence and translation start codon (AUG), are shown. Numbering at the right of each panel is relative to the start oftrpP transcription.

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ture, we used RNase V1 (specific for double-stranded RNA) asa probe to determine whether TRAP interacted with this trip-let. RNase V1 cleavage of G51 within the second repeat wasobserved in the absence of TRAP, whereas bound TRAP pro-tected this residue from RNase V1 cleavage (data not shown).Because RNA structure is known to inhibit TRAP binding totriplet repeats (8, 11, 29, 37), it is likely that breathing of thisstructure allows TRAP interaction with this repeat. Thus, thefootprint results establish that TRAP interacts with all ninerepeats that surround and overlap the trpP S-D sequence andtranslation initiation region. Because the TRAP-RNA cocrys-tal structure shows TRAP interacting with 11 repeats (1), wepresume that TRAP can simultaneously interact with all 9repeats in the trpP transcript.

TRAP inhibits TrpP synthesis by blocking ribosome bind-ing. The position of the TRAP binding target in the trpPtranscript suggested a model in which bound TRAP wouldblock ribosome access to the trpP ribosome binding site. Wecarried out TRAP and 30S ribosomal subunit toeprint exper-iments to test this prediction. The presence of bound TRAP ora 30S ribosomal subunit would block primer extension by re-verse transcriptase, resulting in a toeprint band at a positionnear the 3� boundary of the bound ribosome or TRAP. Thetoeprint results are presented in Fig. 5 and are summarized inFig. 4. Prominent tryptophan-dependent TRAP toeprints wereobserved at positions G85, A96, and U102, corresponding topositions just downstream from repeat 7, within repeat 8, andjust downstream from repeat 9, respectively. An additionalTRAP-dependent toeprint band at U12 was also observed.Since stable RNA secondary structures are capable of blockingextension by reverse transcriptase (e.g., see reference 14), itappears that bound TRAP promotes formation of an RNAstructure near the 5� end of the transcript used in this analysis.In the case of 30S ribosomes, a cluster of three consecutivetRNAfMet-dependent toeprint bands was observed that cen-tered at U99 (Fig. 5). We also carried out toeprint experimentsto determine whether TRAP could inhibit ribosome binding.When TRAP was bound to the trpP transcript prior to theaddition of ribosomes and tRNAfMet, the TRAP toeprintswere observed while the ribosome toeprints were not. These

results demonstrate that bound TRAP inhibits ribosome bind-ing. We also observed a prominent toeprint in all lanes at C54,which is just downstream from the second triplet repeat. Thisresult provides additional evidence for an RNA structure ex-tending from G18 through C54.

Since the toeprint results indicated that bound TRAP com-petes with ribosomes for binding to the trpP transcript, RNA-directed cell-free translation experiments using a TRAP-defi-cient B. subtilis S-30 extract were performed to determinewhether TRAP could inhibit synthesis of a TrpP-green fluo-rescent protein (GFP) fusion peptide from a preexistingmRNA template. This fusion was constructed such that it didnot contain any tryptophan codons, so that in vitro translationcould be performed in the absence or presence of tryptophan.A major protein species that was dependent on the addition ofthe trpP�-�gfp transcript was produced (Fig. 6). No translationproduct corresponding to the fusion peptide was observedwithout the addition of the trpP�-�gfp transcript. In the pres-ence of tryptophan, addition of increasing concentrations ofTRAP to the translation system resulted in a correspondingdecrease in the level of TrpP-GFP synthesis. Inhibition oftranslation was not observed in the absence of added trypto-phan and/or TRAP. In conjunction with the footprint andtoeprint results described above, the cell-free translation ex-periments demonstrated that TRAP binding to the trpP mes-sage inhibits TrpP synthesis by blocking ribosome binding.

DISCUSSION

TRAP-mediated regulation of the trpEDCFBA operon.TRAP binding to the nascent trp leader transcript plays acentral role in regulating expression of the trpEDCFBA operonby transcription attenuation (3, 9, 22, 26, 38) and translationcontrol mechanisms (14, 22, 24, 40). These dual TRAP-depen-dent regulatory mechanisms result in approximately 2,000-foldregulation of the trpEDCFBA operon. When cells are grownunder conditions of tryptophan excess, TRAP would be acti-vated and most likely bind to the message as it is being syn-thesized. In most cases this would promote termination in theleader region (transcription attenuation); however, since ter-

FIG. 4. Summary of the trpP footprint and toeprint results. This figure is adapted from the data presented in Fig. 3 and 5. The composite RNaseT1, RNase T2, and RNase A footprint shows the residues in which cleavage was reduced (�), enhanced (�), or unaffected by bound TRAP ( • ).Residues that were not cleaved in the absence or presence of bound TRAP are indicated with an asterisk. Positions of TRAP and 30S ribosomalsubunit toeprints are indicated by vertical arrows and inverted arrowheads, respectively. The positions of the trpP S-D sequence and AUG startcodon are underlined. Triplet repeats 1 through 9 are shown in parentheses. An inverted repeat that is capable of forming an RNA secondarystructure is indicated with horizontal arrows. Numbering is from the start of trpP transcription.

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mination is never 100% efficient, in some instances RNA poly-merase will escape termination despite TRAP binding. Thiswould result in a TRAP-bound readthrough transcript thatwould sequester the trpE S-D sequence in the trpE S-D block-ing hairpin (14). Inhibition of translation might also occurwhen cells are initially grown under limiting tryptophan con-ditions. In this case, a relatively high percentage of TRAPmolecules would not be activated, resulting in transcriptionreadthrough. Eventually, either by synthesis or transport, asufficient level of tryptophan would build up in the cell andactivate TRAP. Tryptophan-activated TRAP would then bindto the trp leader and promote formation of the trpE S-D block-ing hairpin (14).

Another protein called anti-TRAP (AT) plays a role inregulating tryptophan biosynthesis and transport. AT is a zinc-

containing protein that antagonizes TRAP activity by compet-ing with TRAP’s RNA-binding surface via direct protein-pro-tein interaction (33–35). Transcription of the AT operon isactivated by uncharged tRNATrp via a T-box antiterminationmechanism (18, 28). Interestingly, translation of AT is alsoregulated by uncharged tRNATrp. A short 10-amino-acidleader peptide coding region containing three consecutive Trpcodons just precedes the AT structural gene. It appears thatlow levels of charged tRNATrp cause the ribosome to stall atone or more of these Trp codons, which increases AT synthesis(13). Since expression of the gene encoding AT responds to theaccumulation of uncharged tRNATrp, B. subtilis regulates tryp-tophan biosynthesis and transport by sensing the levels of bothtryptophan and uncharged tRNATrp in the cell.

TRAP-mediated translation control of trpP and pabA. Aputative TRAP binding site was identified in the trpP transcriptthat contained as many as nine triplet repeats (five GAG, threeUAG, and one AAG) (Fig. 1) (27). While the central fiverepeats in the trpP transcript are separated by optimal 2-ntspacers, the first, second, seventh, and eighth spacers contain10, 6, 14, and 1 nt, respectively. Our footprint results indicatethat all nine of these repeats are involved in TRAP-trpP RNAinteraction (Fig. 3 and 4). However, TRAP toeprints were

FIG. 5. TRAP and 30S ribosomal subunit toeprint analysis of trpPRNA. The presence of TRAP, tryptophan (Trp), and/or 30S ribosomalsubunits plus tRNAfMet (30S Rib) is shown at the top of each lane.TRAP was added to the reaction mixture corresponding to the right-most lane prior to the addition of 30S ribosomal subunits andtRNAfMet. Arrows indicate bands corresponding to TRAP and 30Sribosomal subunit toeprints. C54 corresponds to an RNA structuraltoeprint in each lane, whereas U12 corresponds to a TRAP-dependentRNA structural toeprint. Positions of the trpP S-D sequence, the AUGinitiation codon, and the triplet repeats (1 to 9) are shown at the left.Sequencing lanes to reveal U, G, C, or A residues are indicated.

FIG. 6. Effect of tryptophan-activated TRAP on RNA-directedcell-free translation of trpP�-�gfp mRNA. A TRAP-deficient S-30 ex-tract was prepared from B. subtilis strain CYBS306. Reactions werecarried out with various concentrations of purified TRAP in the ab-sence (�) or presence (�) of trpP�-�gfp transcript and/or 1 mM tryp-tophan (Trp). (A) TrpP-GFP translation products analyzed by SDS-polyacrylamide gel electrophoresis. The position of the full-lengthfusion protein is shown with an arrow. (B) Relative level of full-lengthTrpP-GFP polypeptide synthesis as a function of TRAP concentration.The level of polypeptide synthesis in the absence of TRAP was set to1.0.

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observed within repeat 8 and following repeats 7 and 9 (Fig. 4and 5), suggesting that TRAP interaction with the eighth andninth repeats is relatively weak; presumably, reverse transcrip-tase was capable of disrupting TRAP interaction with thesetwo repeats. Perhaps the suboptimal spacing between repeats 7and 8 (14 nt) and between repeats 8 and 9 (1 nt) reduces theaffinity of TRAP for the last two triplets. The position of thetrpP TRAP binding site suggested a model in which boundTRAP would block ribosome binding. Our toeprint (Fig. 5)and in vitro translation (Fig. 6) results demonstrate that boundTRAP inhibits TrpP synthesis by blocking ribosome binding.

Since the trpP translation control mechanism is similar towhat was previously identified for pabA (16, 41), we comparedthe extent of TRAP-mediated translation control of the twogenes and found that TRAP exhibited much tighter control ofTrpP synthesis (Table 2). One reasonable explanation for theless-extensive control of pabA expression is to allow somePabA synthesis in the presence of tryptophan-activated TRAPto maintain folic acid biosynthesis. Despite the considerabledifference in TRAP-dependent translation control of these twogenes, results from our gel mobility shift (Fig. 2) and filter-binding studies indicate that the affinity of TRAP for these twotranscripts is similar. These findings imply that the two trans-lation initiation regions are designed such that bound TRAPhas a greater effect on translation initiation for trpP. Whatmight be responsible for the difference in TRAP-dependentregulation of these two genes?

The TRAP binding targets in pabA (seven GAG, one UAG,and one AAG) and trpP (five GAG, three UAG, and oneAAG) each contain nine repeats. While the optimal spacingbetween repeats is 2 nt, several of the spacers in each transcriptcontain suboptimal spacing. It was previously pointed out thatthe trpP transcript contains five consecutive repeats with opti-mal spacing that overlap and surround its S-D sequence. Incomparison, the pabA transcript contains two stretches of fourrepeats with optimal spacing, one of which overlaps and sur-rounds its S-D sequence (27). Of particular interest is thefinding that the last two repeats in the trpP transcript are withinthe coding region, whereas all nine repeats in pabA are up-stream from the start codon. Perhaps the relative arrangementof repeats in these two transcripts with respect to their cognateS-D sequences and translation initiation regions is at leastpartly responsible for TRAP having tighter control over trpPexpression. Thus, extending the TRAP binding site into thecoding region may be more effective at inhibiting ribosomebinding.

Unlike the case for attenuation, the timing of TRAP bindingdoes not appear to be critical for translation inhibition. In-stead, translation control of pabA and trpP involves a compe-tition between TRAP and ribosome binding. TRAP would onlyinhibit translation while it is bound to a transcript. In theabsence of bound TRAP, a ribosome could bind and initiatetranslation. Once the ribosome clears the translation initiationregion, either TRAP or another ribosome could bind to thetranscript. Thus, a difference in the relative association ordissociation rates of TRAP for the two transcripts could con-tribute to the difference in translation inhibition that was ob-served for these two genes.

It is also possible that the gene arrangement of the twooperons contributes to differences in TRAP-mediated control.

pabA is the second gene in the folate operon, with pabB justupstream. Interestingly, the pabB translation stop codon lieswithin the pabA S-D sequence (Fig. 1). Since only two tripletrepeats in the pabA TRAP binding site lie downstream fromthe pabB stop codon, it is possible that translation of pabBresults in ribosome-mediated displacement of bound TRAP.This would result in a pabA S-D sequence that is transientlyfree of bound TRAP. Thus, ribosome-mediated displacementof bound TRAP might allow translation initiation of pabA.Since trpP is a single-gene operon, ribosome-mediated dis-placement of bound TRAP would not be a factor.

A putative TRAP binding site was also identified that over-laps the S-D sequence and translation initiation region of ycbK,a gene of unknown function (Fig. 1) (28). Thus, it is likely thatTRAP regulates YcbK synthesis by a translation control mech-anism similar to that of pabA and trpP. It is interesting that thestop codon for rtpA, the gene that encodes AT, overlaps theycbK S-D sequence. However, in this case only one of thetriplet repeats is upstream of the S-D sequence, while six arepresent in the ycbK coding region (Fig. 1). Thus, one mightpredict that ribosome-mediated displacement of bound TRAPwould be less pronounced than for pabA, while the repeats inthe coding sequence may increase the effectiveness of boundTRAP in blocking ribosome binding.

It is well established that RNA structure can inhibit TRAPbinding (8, 11, 29, 37). In contrast, TRAP interaction with the5� stem-loop (5�SL) that forms at the extreme 5� end of the trpleader transcript increases the affinity of TRAP for the trpleader by an unknown mechanism (15). Our footprint andtoeprint results, combined with computer modeling, revealedan RNA secondary structure extending from nt 18 to 54 of thetrpP leader transcript. The first triplet repeat is present in theloop of the hairpin, while the second repeat is in the 3� half ofthe stem (Fig. 4). Interestingly, the trp leader 5�SL contains aGAG in the loop of the hairpin and an AAG in the 3� half ofthe stem (15, 32); however, neither of these triplets is part ofthe 11-repeat TRAP-binding target identified by footprinting(7). A systematic mutational analysis of the 5�SL indicated thatthe overall structure was important for TRAP-dependent reg-ulation. While changing the GAG in the loop of the 5�SL toGUG had only a small effect on TRAP-mediated regulation,mutating the AAG in the stem to AAC resulted in a consid-erable reduction in TRAP’s ability to regulate trp operon ex-pression (32). Thus, the 5�SL contains both structural andsequence elements that participate in TRAP binding. Perhapsin addition to contributing two triplet repeats, the trpP leaderhairpin may provide a structural element that participates inTRAP interaction. Since the pabA transcript does not containa similar structure, it is possible that the trpP RNA hairpincontributes to the difference in TRAP-mediated regulation ofthese two genes.

ACKNOWLEDGMENTS

We thank Paul Gollnick and Charles Yanofsky for providing bacte-rial strains and Paul Lovett for 30S ribosomal subunits.

This work was supported by grant GM52840 from the NationalInstitutes of Health.

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