trp RNA-Binding Attenuation Protein-59 Stem-Loop RNA ...weijenlin/Bio560/TRP operon.pdfExpression of the Bacillus subtilis tryptophan biosynthetic genes is regulated in response to

JOURNAL OF BACTERIOLOGY,0021-9193/00/$04.0010

Apr. 2000, p. 1819–1827 Vol. 182, No. 7

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

trp RNA-Binding Attenuation Protein-59 Stem-Loop RNA InteractionIs Required for Proper Transcription Attenuation Control

of the Bacillus subtilis trpEDCFBA OperonHANSEN DU,† ALEXANDER V. YAKHNIN, SUBRAMANIAN DHARMARAJ,‡ AND PAUL BABITZKE*

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

Received 25 October 1999/Accepted 10 January 2000

The trp RNA-binding attenuation protein (TRAP) regulates expression of the Bacillus subtilis trpEDCFBAoperon by a novel transcription attenuation mechanism. Tryptophan-activated TRAP binds to the nascent trpleader transcript by interacting with 11 (G/U)AG repeats, 6 of which are present in an antiterminatorstructure. TRAP binding to these repeats prevents formation of the antiterminator, thereby promoting for-mation of an overlapping intrinsic terminator. A third stem-loop structure that forms at the extreme 5* end ofthe trp leader transcript also plays a role in the transcription attenuation mechanism. The 5* stem-loopincreases the affinity of TRAP for trp leader RNA. Results from RNA structure mapping experiments demon-strate that the 5* stem-loop consists of a 3-bp lower stem, a 5-by-2 asymmetric internal loop, a 6-bp upper stem,and a hexaloop at the apex of the structure. Footprinting results indicate that TRAP interacts with the 5*stem-loop and that this interaction differs depending on the number of downstream (G/U)AG repeats presentin the transcript. Expression studies with trpE*-*lacZ translational fusions demonstrate that TRAP-5* stem-loop interaction is required for proper regulation of the trp operon. 3* RNA boundary experiments indicate thatthe 5* structure reduces the number of (G/U)AG repeats required for stable TRAP-trp leader RNA association.Thus, TRAP-5* stem-loop interaction may increase the likelihood that TRAP will bind to the (G/U)AG repeatsin time to block antiterminator formation.

Expression of the Bacillus subtilis tryptophan biosyntheticgenes is regulated in response to changes in the intracellularlevel of tryptophan by the trp RNA-binding attenuation protein(TRAP) (4, 16). The trpEDCFBA operon is regulated byTRAP-mediated transcription attenuation (5, 10, 17, 19, 24,25) and translational control mechanisms (14, 19, 22). TRAPalso regulates expression of the unlinked trpG gene at thetranslational level (8, 15, 30). TRAP exists as a complex con-sisting of 11 identical subunits arranged in a single ring termedthe b-wheel (1, 3). Tryptophan cooperatively activates TRAPby binding between every adjacent TRAP subunit (3, 6).

The 203-nucleotide untranslated trp operon leader transcriptcan fold into three distinct RNA secondary structures thatparticipate in transcription attenuation (Fig. 1). When TRAPis activated by tryptophan, 11 KKR motifs that outline theperiphery of the TRAP complex can bind to 11 closely spaced(G/U)AG repeats present in the nascent trp leader transcript,thereby wrapping the RNA around the periphery of the TRAPcomplex (2, 8, 31). TRAP binding blocks formation of theantiterminator since six of the (G/U)AG repeats are presentwithin this RNA structure (5, 8). Thus, TRAP binding pro-motes formation of the overlapping intrinsic terminator whichresults in transcription termination before RNA polymerasecan reach the trp operon structural genes. In the absence ofTRAP binding, formation of the antiterminator permits tran-scription of the entire operon (5).

While it is not known how TRAP initially interacts with thenascent trp leader transcript, the interaction must occur quicklyto prevent formation of the antiterminator structure. Duringattenuation regulation of the Escherichia coli trp operon, tran-scriptional pausing allows the regulatory ribosome to bind tothe leader transcript at an appropriate time (20). Since leaderpeptide synthesis is not involved in transcription attenuation ofthe B. subtilis trp operon, nor has RNA polymerase pausingbeen demonstrated to play a role in this regulatory mechanism,we were interested in determining if any factor besides TRAPand the (G/U)AG repeats were involved in TRAP interactionwith the nascent trp leader transcript.

We recently demonstrated that, in addition to the antiter-minator and terminator, an RNA structure predicted to format the extreme 59 end of the nascent trp leader transcriptparticipates in the transcription attenuation mechanism (28).Deletion or disruption of this putative structure resulted in adramatic increase of trp operon expression in vivo and in-creased transcriptional readthrough in vitro. This previousstudy also demonstrated that the 59 stem-loop functions pri-marily in TRAP-dependent regulation of the trp operon andthat overexpression of TRAP suppressed the defect associatedwith the 59 stem-loop deletion. Moreover, we showed that thepresumed 59 structure increased the affinity of TRAP for trpleader RNA (28). Thus, it was possible that the 59 stem-loopparticipated in the attenuation mechanism by interacting withTRAP.

In the present study we determined the secondary structureof the 59 stem-loop and found that TRAP interacts with thisstructure. We also established that the 59 stem-loop reducesthe number of (G/U)AG repeats required for stable TRAP-trpleader RNA association and that the TRAP-59 stem-loop in-teraction differs depending on the number of downstream (G/U)AG repeats that are present in the transcript. Our results

* 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: [email protected].

† Present address: Department of Biology, MS008, Brandeis Uni-versity, Waltham, MA 02454.

‡ Present address: Ambion, Inc., Austin, TX 78744.

1819

suggest that the TRAP-59 stem-loop interaction increases theprobability that TRAP will bind to the (G/U)AG repeats be-fore the antiterminator can form, thereby increasing the like-lihood that transcription termination occurs before RNA poly-merase can reach the trp operon structural genes.

MATERIALS AND METHODS

Bacterial strains and plasmids. All of the B. subtilis strains used in this studyare listed in Table 1. The plasmids pTZ18U (Stratagene) and pPB77, pPB78,pPB82, and pPB83 (8) have been described. Plasmid pPB310 contains nucleo-tides 32 to 111 of the B. subtilis trp leader region and was constructed by PCR.The resulting PCR product was digested with EcoRI and BamHI and subclonedinto the EcoRI and BamHI sites of the pTZ18U polylinker. Plasmid pHD55,which contains nucleotides 1 to 36 of the B. subtilis trp leader, was also con-structed by PCR. In this case the PCR product was digested with EcoRI and KpnIand ligated into the EcoRI and KpnI sites of the pTZ18U polylinker. PlasmidpHD68 was constructed by digesting pHD55 with EcoRI and treating it withmung bean nuclease to remove the cohesive ends followed by self-ligation.

Plasmid pHD34 contains the trp promoter and nucleotides 6 to 203 D(11 to 15)of the trp leader. This plasmid was constructed by a two-step process usingoverlap extension PCR. The final PCR product was digested with EcoRI andHindIII and subcloned into the EcoRI and HindIII sites of PTZ18U. pHD40carries the trp promoter and a mutant trp leader in which nucleotides 6 to 9 werereplaced with a T residue, while pHD46 carries the trp promoter and a leadercontaining nucleotides 16 to 203 D(11 to 115). Both of these plasmids wereconstructed in the same manner as pHD34. The B. subtilis integration vector,ptrpBG1-PLK, used for the generation of trpE9-9lacZ translational fusions wasdescribed previously (22). The plasmids pHD52, pHD53, and pHD54, whichcontain trpE9-9lacZ fusions, were constructed by subcloning the trp promoter andleader region from pHD34, pHD40, and pHD46 into the EcoRI and HindIIIsites of the ptrpBG1-PLK polylinker, respectively. The three plasmids pHD52,pHD53, and pHD54 were linearized with SalI and separately integrated into theamyE locus of B. subtilis W168. The resulting strains are PLBS138, PLBS139, andPLBS140.

b-Galactosidase assay. Cells were cultured in minimal Spizizen salts medium(27) containing 0.2% acid-hydrolyzed casein, 0.2% glucose, and 5 mg of chlor-amphenicol per ml in the presence or absence of 50 mg of tryptophan per ml.Cells were harvested in mid-exponential phase, and cell suspensions were pre-pared as previously described (28). b-Galactosidase activity was subsequentlyassayed by the method of Miller (23).

In vitro transcription. Gel-purified transcripts used in this analysis were syn-thesized by using the Ambion MEGAscript in vitro transcription kit. Templatesconsisted of various plasmids that had been linearized with BamHI or HindIII.59-End-labeled RNAs were generated by treating in vitro-generated transcriptswith calf intestinal phosphatase and subsequently with polynucleotide kinase and[g-32P]ATP. The unlabeled and labeled RNA was gel purified as previouslydescribed (14).

Gel mobility shift assay. The binding affinity between TRAP and trp leaderRNA was estimated by using gel mobility shift assays by modifying a previouslypublished procedure (28). TRAP was purified as described earlier (5). Tran-scripts used in the analysis were generated from pPB77 (wild type), pPB310 (59stem-loop deletion), or pHD68 (59 stem-loop only) that had been linearized withBamHI. Binding reactions (8 ml) containing 0.2 nM 59-end-labeled RNA, variousconcentrations of TRAP (TRAP excess), 1 mM tryptophan in 50 mM Tris-acetate (pH 8.0), 4 mM magnesium acetate, 5 mM dithiothreitol, 10% glycerol,0.2 mg of E. coli tRNA per ml, 0.1 mg of xylene cyanol per ml, and 400 U ofRNasin (Promega) per ml were incubated at 25°C for 20 min. Aliquots ofreaction mixtures were fractionated through native polyacrylamide gels contain-ing 375 mM Tris-HCl (pH 8.8), 5% glycerol, and 1 mM EDTA. Electrophoresiswas performed at room temperature in running buffer containing 25 mM Tris-glycine (pH 8.3) and 1 mM EDTA. Gels were dried, and the bound and freeRNA bands were quantified by using a PhosphorImager (Molecular Dynamics)and the ImageQuant software package. Modifications of the standard reactionare described in the text or the appropriate figure legend. The binding data werefit to the simple binding equation: RNAb 5 a[TRAP]f/(Kd 1 [TRAP]f), where ais the maximal fraction of bound RNA (RNAb) that is approximately equal to 1;Kd is defined as the concentration of free protein, [TRAP]f, at which the RNAbreaches 50% saturation; RNAb is the fraction of RNA bound between 0 and 1;and [TRAP]f is the concentration of free TRAP 11-mer which was assumed to bethe concentration of total TRAP added since the total TRAP concentration wasin at least 12-fold molar excess over RNA.

RNA structure mapping. RNA structures were predicted by using theMFOLD program (29, 32). RNA structure mapping using unlabeled transcriptsfollowed previously published procedures (14). The unlabeled transcripts used inthis analysis were generated from pPB83 linearized with HindIII as template.Titrations of RNases and chemical reagents were routinely performed to deter-mine the amount of each reagent that would prevent multiple cleavages orchemical modifications in any one transcript so that we could minimize thepotential of secondary rearrangements in short RNA segments. RNA sampleswere partially digested with RNase T1 (Gibco-BRL) or RNase V1 (Pharmacia)and recovered as described earlier (14). CMCT and DMS modification reactions,as well as the subsequent recovery of RNA samples, followed a previouslypublished procedure (14). RNA samples were resuspended in primer extensionbuffer and hybridized to a g-32P-end-labeled primer, and the primers wereextended with Moloney murine leukemia virus (MMLV) reverse transcriptase(U.S. Biochemicals) as described elsewhere (14). After 10 min at 42°C, reactionswere terminated by the addition of 3 ml of standard sequencing stop solution.Samples were fractionated through 6% denaturing polyacrylamide gels. Controlsequencing reactions were carried out using the same plasmids and end-labeledprimer as described above.

59-end-labeled RNA (see above) generated from various templates (pPB77,pPB78, pPB82, pPB83, or pHD68 digested with BamHI) was renatured byheating at 95°C for 1 min, followed by a 10-min incubation at 37°C. RNA wasdigested with 0.07 U of RNase V1 per ml for 10 min at 37°C in 40 mM Tris-HCl(pH 8.0)–250 mM KCl–4 mM MgCl2 (TKM buffer). Samples were fractionatedthrough 6% denaturing polyacrylamide gels. The G sequencing ladder was gen-erated by partial RNase T1 digestion under denaturing conditions as describedpreviously (11). Alkali digestion ladders were prepared as described elsewhere(13) from the same end-labeled transcripts.

FIG. 1. Nucleotide sequence of the B. subtilis trp leader transcript showingthe 59 stem-loop and the mutually exclusive antiterminator and terminator struc-tures. Boxed nucleotides mark overlapping segments of the competing secondarystructures. The (G/U)AG repeats known to be involved in TRAP-RNA recog-nition are indicated by boldface type. Numbering is from the start of transcrip-tion. RNA secondary structure predictions were performed using MFOLD (29,32). Note that the 59 stem-loop is modified from the structure predicted byMFOLD due to the RNA secondary structure mapping data obtained during thecourse of these studies.

TABLE 1. B. subtilis strains used in this study

Strains Genotypea Source orreference

W168 Prototroph BGSCc

PLBS44 amyE::[trpP(2412 to 1203)trpE9-9lacZ Cmr]

28

PLBS104 amyE::[trpP(2412 to 1203)D(13 to 132)trpE9-9lacZ Cmr]

28


This study

PLBS139 amyE::[trpP(2412 to 1203)D(16 to 19)Tb trpE9-9lacZ Cmr]

This study


This study

a trpP denotes the trp promoter. Prime indicates truncation of the gene. 2412to 1203 indicates the DNA fragment containing the trp promoter and neighbor-ing regions that was incorporated relative to the transcription start site. “D”designates the portion of the leader region that was deleted.

b T was substituted for nucleotides 6 to 9.c BGSC, Bacillus Genetic Stock Center, Ohio State University, Columbus,

Ohio.

1820 DU ET AL. J. BACTERIOL.

3*-boundary analysis. The 39-boundary analysis followed a published proce-dure (11). 59-end-labeled transcripts (see above) generated from pPB77 (wild-type trp leader) or pPB310 (59 stem-loop deletion trp leader) were treated withalkali to generate an RNA ladder. Then, 100-ml RNA samples (10 pmol) wereincubated for 5 min at 95°C in alkaline hydrolysis buffer (100 mM NaHCO3-Na2CO3 [pH 9.0]–2 mM EDTA–0.5 mg of E. coli tRNA per ml) and thenrecovered by ethanol precipitation. Hydrolyzed RNAs were mixed with 50 mg ofTRAP and incubated at 25°C for 20 min in TKM buffer. The reaction mixtureswere fractionated through 6% native polyacrylamide gels. Bound and unboundtranscripts were visualized by autoradiography, excised from the gel, and subse-quently eluted from the gel. RNAs were ethanol precipitated and fractionatedthrough 6% denaturing polyacrylamide gels. RNase T1 and alkali digestionladders of the same 59-end-labeled transcripts were used as molecular size stan-dards.

RESULTS

Gel mobility shift analysis of TRAP and trp leader RNA.Results from previous in vivo experiments demonstrated thatoverexpression of mtrB, the gene encoding TRAP (17), sup-pressed the defect associated with deletion of the 59 stem-loop(28). Using gel mobility shift assays we further showed that the59 stem-loop increases the affinity of TRAP for trp leader RNAapproximately fivefold (28). In the previous study (28) we ob-served a TRAP-dependent band that migrated just behind thefree RNA. We assumed that this band resulted from TRAP-trpleader RNA complex dissociation soon after loading the gel.When we repeated the analysis using a modified gel shift pro-cedure (see Materials and Methods) the presence of this bandwas eliminated, confirming our previous assumption. As waspreviously observed (28), the presence of the 59 structure intranscripts that contained all 11 (G/U)AG repeats increasedthe affinity of TRAP for trp leader RNA (Fig. 2). Binding to thewild-type trp leader transcript was detectable at 2.5 nM TRAPand saturated at approximately 320 nM TRAP (Fig. 2A). Withthe 59 stem-loop deletion transcript, comparable binding wasdetected at 5 nM TRAP but did not reach saturation even at aconcentration of 1.28 mM TRAP (Fig. 2B). In each case weobserved a prominent shifted complex. Note that we also ob-served two additional shifted complexes for each of these tran-scripts. One of these complexes is shown (p), while the otherextremely faint complex is not. Note that these complexes werenot observed in our previous study (28). While the most prom-inent shifted species probably consists of complexes containingone TRAP 11-mer bound to a single trp leader transcript, thecomposition of the other shifted species is not known. We fitthese data to a simple binding equation by using nonlinearleast-squares analysis. This method yielded estimated Kd val-ues of 26 6 5 nM TRAP for the wild-type transcript and 280 650 nM for the 59 stem-loop deletion transcript. The smalldifference in these values from those observed previously (28)probably reflects the different binding and gel-running condi-tions used in the current study.

The finding that the 59 stem-loop increased the affinity ofTRAP for trp leader RNA approximately 10-fold suggestedthat the 59 stem-loop interacted with TRAP. When we per-formed gel shift experiments with transcripts derived fromplasmid pHD68 that only contained the 59 stem-loop, we didnot detect any evidence of TRAP binding (data not shown).We also performed RNA competition experiments with wild-type trp leader transcripts and transcripts that only containedthe 59 stem-loop. While the unlabeled wild-type trp leadertranscript was able to compete for TRAP binding to labeledwild-type and 59 stem-loop deletion trp leader transcripts, thetranscript that only contained the 59 stem-loop only competedaway the higher-shifted complexes (p) (data not shown). Sincethe RNA that only contained the 59 stem-loop was an ineffec-tive competitor, these results suggest that TRAP–59 stem-loop

RNA interaction does not involve the KKR motifs known tointeract with the (G/U)AG repeats (2, 31).

5* stem-loop structure mapping. To determine if the pre-dicted 59 structure actually formed in the trp leader transcript,we probed the structure of a transcript containing the first 68nucleotides of the trp leader in vitro with structure-specificenzymatic and chemical reagents. This transcript contained thepredicted 59 stem-loop and the first six (G/U)AG repeatsknown to interact with TRAP, as well as four upstream anddownstream nucleotides derived from the vector. Note thatcomputer predictions indicated that these additional residuesdo not interfere with 59 stem-loop formation. trp leader tran-scripts were subjected to partial digestion or chemical modifi-cation using RNase T1, RNase V1, DMS, or CMCT. The sitesof nuclease cleavage or chemical modification were mapped byprimer extension using an end-labeled primer and MMLVreverse transcriptase. Cleavage or chemical modificationwould give rise to a primer extension product one nucleotideshorter than the corresponding band in the sequencing lane.

The results of the structure mapping experiments are shownin Fig. 3 and summarized in Fig. 4. The computer-predictedstructure of the 59 stem-loop is identical to the experimentallydetermined structure except that U5 and A29 are predicted topair as are A16 and U21. RNase T1 cleaves following unpairedG residues. We observed prominent RNase T1 cleavage fol-lowing the G residues at positions 18, 20, 38, and 42, indicatingthat these residues are single stranded. Note that bands cor-

FIG. 2. Gel mobility shift analysis of TRAP complexed with wild-type or 59stem-loop deletion trp leader transcripts. 59-end-labeled trp leader transcripts(0.2 nM) were incubated with 1 mM tryptophan and the concentration of TRAPindicated at the bottom of each lane (nanomolar). Each transcript contained the11 (G/U)AG repeats between nucleotides 36 and 91. Bands corresponding tofree (F) and bound (B or p) RNA are indicated on the left. (A) Wild-type trpleader transcripts. (B) 59 stem-loop deletion trp leader transcripts.

VOL. 182, 2000 TRAP-59 STEM-LOOP RNA INTERACTION 1821

responding to the G residues at positions 2, 7, 22, 24, and 31were not detected, suggesting that these residues were basepaired (Fig. 3 and 4). With the exception of G7, these resultsare consistent with the computer predicted secondary struc-ture. RNase V1 is generally specific for base-paired residues;however, this enzyme does not cleave all paired residues, andit sometimes cleaves the first few bases in a single-strandedRNA segment that is adjacent to an RNA duplex (26). Weobserved prominent RNase V1 cleavage following A1, U4,U11, A12, and U23, as well as weak RNase V1 cleavage fol-lowing C3, A10, G24, C32, and A33, suggesting that theseresidues are base paired (Fig. 3 and 4). These results areconsistent with the predicted 59 stem-loop structure. Note thatthe cleavage of A1 and A33 is likely due to their positionimmediately adjacent to the lower stem of the structure.

To determine the structure of the 59 stem-loop more pre-cisely, chemical modification experiments with DMS andCMCT were carried out. DMS methylates N1 of adenine andN3 of cytosine when the residues are single stranded, whereas

CMCT modifies unpaired G and U residues at the N1 and N3

positions, respectively. These DMS- and CMCT-modified res-idues are unable to serve as templates for reverse transcriptase.We observed DMS signals at the A and C residues correspond-ing to positions 1, 6, 8, 9, 16, 17, 19, 28, 29, 30, 32, 33, 34, 37,39, and 40, suggesting that these residues are single stranded.However, the relatively weak DMS signals at positions 1, 30,and 32 suggest that these residues can be paired or unpaired.The absence of DMS modification of the remaining A and Cresidues suggests that these residues are base paired. With theexception of A16 and A29, these results are consistent with thepredicted structure (Fig. 3 and 4). We observed prominentCMCT signals at the U and G residues corresponding to po-sitions 5, 7, 18, 20, 21, 35, 36, 38, and 41, indicating that theseresidues are single stranded. The absence of CMCT modifica-tion of the remaining U and G residues suggests that thesenucleotides are base paired (Fig. 3 and 4). With the exceptionof G7, which was not cleaved by RNase T1, the CMCT resultsare consistent with the other reagents tested. Taken together,the results of the structure mapping experiments are consistentwith the structure shown in Fig. 4, although it appears that thelower stem is relatively unstable. The structure that we deter-mined differs from the predicted structure by two base pairs.The predicted U5-A29 and the A16-U21 base pairs were notdetected in the 59 stem-loop secondary structure, suggestingthe existence of a larger asymmetric internal loop and hairpinloop, respectively (Fig. 4). When taken together, our structuremapping results indicate that the 59 stem-loop consists of a3-bp lower stem, a 5-by-2 asymmetric internal loop, a 6-bpupper stem, and a hexaloop at the apex of the structure.

TRAP interacts with the 5* stem-loop. Our gel shift analysisindicated that the 59 stem-loop increases the affinity of TRAPfor trp leader RNA but provided little evidence that TRAPinteracts with the 59 structure. We performed TRAP-trp leaderRNA footprint experiments to determine if TRAP interactswith the 59 stem-loop (Fig. 5). We used the same in vitro-generated trp leader transcript (positions 1 to 68), chemicaland enzymatic probes, and 59-end-labeled primer used for the

FIG. 3. 59 stem-loop structure mapping. RNA containing nucleotides 1 to 68of the trp leader transcript was used in this analysis (Fig. 1). trp leader RNA wastreated with RNase T1, RNase V1, DMS, or CMCT. Residues that were cleavedby RNase T1 or RNase V1 or modified by DMS or CMCT were detected byprimer extension by using MMLV reverse transcriptase. The mock-treated con-trol lane without enzymatic or chemical treatment is indicated. Note that thebands observed in the treated lanes are one nucleotide shorter than the corre-sponding bands in the A, C, G, or U sequencing lanes. The positions of thenucleotides corresponding to the lower stem, the internal loop, the upper stem,the hexaloop, and the first (G/U)AG repeat (UAG) are indicated at the right.Numbering at the left corresponds to the DNA sequencing ladder and is from thestart of transcription.

FIG. 4. Summary of the 59 stem-loop structure mapping results. This figure isadapted from the data presented in Fig. 3. Positions of cleavage by the single-stranded probe RNase T1 are indicated by arrows. Positions of cleavage by thedouble-stranded probe RNase V1 are indicated by arrowheads. Positions of RNAmodification using the single-stranded probe DMS (circles) or CMCT (squares)are also indicated. Filled arrowheads, circles, or squares indicate strong modifi-cation or cleavage, whereas open arrowheads, circles, or squares indicate weakmodification or cleavage. Numbering is from the start of transcription.


structure-mapping experiments (see above). The cleavage pat-tern with RNase V1 was dramatically altered when TRAP wasbound to the trp leader transcript. Bound TRAP reduced orprevented RNase V1 cleavage at every 59 stem-loop residuethat was cleaved in the absence of TRAP (Fig. 5). Since RNaseV1 is generally specific for double-stranded RNA, these resultssuggested that TRAP bound to the 59 structure and preventedcleavage. In sharp contrast, the RNase T1 cleavage patternwithin the 59 stem-loop was only slightly altered when TRAPwas bound to the transcript (Fig. 5). Interestingly, the RNaseT1 cleavage pattern suggests that the GAG sequence in theloop of the 59 structure does not interact with a TRAP KKRmotif (Fig. 4 and 5). Previous results demonstrated that both Gresidues in GAG repeats are strongly protected from RNaseT1 cleavage by bound TRAP (8, 15). This finding is consistentwith a previous in vivo study where it was determined thatchanging this sequence to GUG had little effect on trp operonexpression (28). Note that, with the exception of the first UAGrepeat, bound TRAP prevented or reduced cleavage of the Gresidues in the (G/U)AG repeats that were previously shownto interact with TRAP (data not shown) (18).

As was observed for RNase V1 cleavage, the DMS andCMCT RNA modification patterns were significantly alteredwhen TRAP was bound to the trp leader transcript. We foundthat bound TRAP protected A8, A9, A19, A30, C32, A34, and

A37 from DMS methylation, whereas bound TRAP enhancedmodification of A1 (Fig. 5). In the case of CMCT, boundTRAP protected G7, G18, G20, U21, and G39 from CMCTmodification, whereas modification of U4 was enhanced whenTRAP was bound. It should be pointed out that the resultswith CMCT and RNase T1 are not in agreement. WhereasTRAP protected G18 and G20 from CMCT modification,bound TRAP did not significantly protect either of these res-idues from RNase T1 cleavage. The reason for this discrepancyis unknown. When taken together, the footprinting results areconsistent with a TRAP-59 stem-loop RNA complex contain-ing the internal loop, the upper stem, the hexaloop, and the 39side of the lower stem. Note that in no case did we detectTRAP binding to the trp leader transcript in the absence oftryptophan (data not shown).

TRAP interaction with the 5* stem-loop is required forproper regulation of the trp operon. The TRAP–59 stem-loopfootprint results suggested that TRAP does not interact withthe 59 side of the lower stem. To determine if the lower stemis important for 59 stem-loop function, we deleted the DNAregion corresponding to the first five nucleotides of the trpleader transcript. We examined B. subtilis strains containingtrpE9-9lacZ translational fusions that were controlled by thewild type (WTtrpL), the 59 stem-loop deletion D(13 to 132),or the D(11 to 15) trp leader and analyzed b-galactosidaseexpression when each strain was grown in the presence orabsence of exogenous tryptophan. We observed minimal ex-pression in the WTtrpL strain PLBS44 grown in the presenceof tryptophan (Table 2). The effect of exogenous tryptophanon the expression of the WTtrpL trpE9-9lacZ fusion can beassessed from the 2Trp/1Trp ratio, which was 260. Compa-rable experiments were performed with the D(13 to 132)strain PLBS104 and the D(11 to 15) strain PLBS138. As waspreviously observed (28), deletion of the entire 59 stem-loopresulted in a dramatic increase in expression, especially whencells were grown in the presence of tryptophan. In this case the2Trp/1Trp ratio was only 23, significantly lower than thatobserved for the WTtrpL strain (Table 2). Interestingly, theexpression levels of the D(11 to 15) strain were similar to thewild-type strain (Table 2), indicating that these residues arenot required for 59 stem-loop function (Table 2). This result isconsistent with the footprint analysis (Fig. 5).

The footprint results presented above also indicated that the59 side of the asymmetric internal loop is involved in TRAP-59stem-loop interaction. We replaced nucleotides 6 to 9(AGAA) of the internal loop with a single U residue. Thepredicted structure of this mutant transcript contained a con-tiguous 12-bp stem without an internal loop (structure notshown). We examined the effect of this trp leader mutation on

FIG. 5. TRAP–59 stem-loop RNA footprint. RNA containing nucleotides 1to 68 of the trp leader transcript was used in this analysis (Fig. 1). trp leader RNAwas treated with RNase T1, RNase V1, DMS, or CMCT in the presence (1) orabsence (2) of bound TRAP. Residues that were cleaved by RNase T1 or RNaseV1 or modified by DMS or CMCT were detected by primer extension by usingMMLV reverse transcriptase. The mock-treated control lane without enzyme orchemical treatment is indicated. Note that the bands observed in the treatedlanes are one nucleotide shorter than the corresponding bands in the A, C, G, orU sequencing lanes. The positions of the nucleotides corresponding to the lowerstem, the internal loop, the upper stem, the loop, and the first (G/U)AG repeat(UAG) are indicated on the right. The numbering on the left corresponds to theDNA sequencing ladder and is from the start of transcription.

TABLE 2. Effect of 59 stem-loop mutations on trpoperon expression

Strain 59 stem-loopmutation

b-Gal activityb

(Miller units) b-Gal ratio(2Trp/1Trp)

1Trp 2Trp

PLBS44 Wild type 0.2 6 0.05 52 6 5 260PLBS104 D(13 to 132) 12 6 0.5 274 6 11 23PLBS138 D(11 to 15) 0.4 6 0.07 75 6 2 188PLBS139 D(16 to 19)

replaced with Ua4.2 6 0.4 190 6 15 45

PLBS140 D(11 to 115) 3.7 6 0.2 152 6 12 41

a Nucleotides 6 to 9 (AGAA) were replaced by U in the trp leader transcript.b b-Galactosidase (b-Gal) activity expressed from the trpE9-9lacZ fusion is

given in Miller units (23).


expression of a trpE9-9lacZ translational fusion (PLBS139).Compared to the wild-type strain, we found that b-galactosi-dase levels increased 20-fold when this strain was grown in thepresence of tryptophan and 4-fold in its absence (Table 2). Inthis case the 2Trp/1Trp ratio was 45, only twofold higher thanthat observed for the strain in which the entire 59 stem-loopwas deleted. This result indicates that the 59 side of the asym-metric internal loop is important for 59 stem-loop function,which again is consistent with the footprint results. We alsoexamined the effect of a deletion that extended from 1 to 15and found that the expression levels in this strain (PLBS140)were similar to those of the 59 stem-loop deletion strain(PLBS139) (Table 2).

The 5* stem-loop reduces the number of (G/U)AG repeatsrequired for tight TRAP-trp leader RNA binding. Our foot-print and gel shift results demonstrated that TRAP interactswith the 59 stem-loop and that this interaction increases theaffinity of TRAP for trp leader RNA. We performed a 39boundary analysis using wild type (nucleotides 1 to 111) and 59stem-loop deletion (nucleotides 32 to 111) trp leader tran-scripts to determine if the 59 stem-loop reduced the number of(G/U)AG repeats that were required for tight TRAP-trp leaderRNA binding. Note that these two transcripts were identical tothose used in the gel shift analysis (Fig. 2). RNAs were 59 endlabeled, hydrolyzed to obtain a ladder of 59-end-labeled tran-scripts, and subsequently mixed with tryptophan-activatedTRAP. Bound and unbound RNAs were separated by nativegel electrophoresis, gel purified, and separated on a standarddenaturing sequencing gel. We observed cutoffs betweenbound and unbound transcripts with both the wild-type and 59stem-loop deletion trp leader transcripts (Fig. 6). The cutoff forthe wild-type trp leader transcript was relatively sharp andoccurred at between seven and eight (G/U)AG repeats, withbound and unbound lanes showing complementary cutoffs andcutons. Under the binding conditions employed here, this re-sult demonstrated that the first six (G/U)AG repeats wererequired for stable TRAP-trp leader RNA complex formationwhen the 59 stem-loop was present in the transcript. However,a small fraction of the transcripts that contained as few as threerepeats was also shifted. Interestingly, the corresponding cutofffor the 59 stem-loop deletion transcript occurred at betweennine and ten (G/U)AG repeats, indicating that the first eight(G/U)AG repeats were required for comparable binding. Inthis case a small fraction of the transcripts that contained asfew as six repeats were also shifted. Note that the short tran-scripts containing fewer than five (G/U)AG repeats in theunbound 59 stem-loop deletion sample were not gel purified inthis experiment (Fig. 6) since previous experiments indicatedthat these transcripts were not gel shifted by TRAP. The re-sults of the 39 boundary analysis demonstrate that the 59 stem-loop structure reduces the number of (G/U)AG repeats re-quired for stable TRAP association.

The nature of the TRAP–5* stem-loop RNA interaction isdependent on the number of downstream (G/U)AG repeats.Results from a previous study demonstrated that the 59 stem-loop functions in the transcription attenuation mechanism thatcontrols expression of the trp operon (28). Furthermore, theresults described above indicate that TRAP interacts with the59 stem-loop and that this interaction increases the affinity ofTRAP for trp leader RNA. Moreover, we found that the pres-ence of the 59 structure reduces the number of (G/U)AGrepeats required for stable TRAP-trp leader RNA association.One possible explanation for these results is that the 59 stem-loop might tether TRAP to the nascent trp leader transcriptsuch that TRAP would be in position to bind to the (G/U)AGrepeats as soon as they are transcribed. A multipartite bind-

ing mechanism such as this might increase the probability thattryptophan-activated TRAP would bind to the trp leaderin time to block antiterminator formation. For this bindingmechanism to have the greatest impact on trp operon expres-sion, one would predict that TRAP–59 stem-loop interactionwould occur in the absence of any downstream (G/U)AG re-peats.

We performed a TRAP-RNA footprint experiment using59-end-labeled trp leader transcripts that contained the 59stem-loop in the absence of any downstream (G/U)AG repeats(nucleotides 1 to 36) to determine if TRAP could interact witha transcript that only contained the 59 structure. The RNase V1cleavage pattern in the absence of TRAP differed from thecleavage pattern when TRAP was present (Fig. 7). We ob-served appreciable RNase V1 cleavage in the presence or ab-sence of TRAP following U11, A12, U23, and G24. Surpris-ingly, cleavage following U25, A26, G31, and C32 was onlyobserved in the presence of TRAP. These results indicate thatTRAP can interact with the 59 stem-loop in the absence of the11 (G/U)AG repeats and that this interaction was transient,resulting in a 59 stem-loop that is more highly structured. Thefact that our gel shift assay was unable to detect a complexbetween TRAP and a transcript that only contained the 59stem-loop (data not shown) is consistent with rapid TRAP-59stem-loop RNA complex dissociation.

FIG. 6. 39 boundary analysis of wild-type and 59 stem-loop deletion trp leadertranscripts. Limited alkaline hydrolysis ladders of 59-end-labeled wild-type (WT)or 59 stem-loop deletion trp leader transcripts were incubated with tryptophan-activated TRAP. TRAP-RNA complexes were separated from unbound RNA ona native gel and subsequently fractionated through a denaturing 6% polyacryl-amide gel (shown). Labels for lanes are as follows: OH2, a limited alkalinehydrolysis ladder; T1, partial RNase T1 digest; B and U, bound and unbound areRNA fragments from the limited alkaline hydrolysis that either bound (B) or didnot bind (U) TRAP. The numbers on the left (wild-type transcript) or right (59stem-loop deletion transcript) indicate the relative positions of the (G/U)AGrepeats, with 1 being closest to the 59 end of the transcript.


We then examined the effect TRAP binding had on 59 stem-loop RNase V1 cleavage patterns when transcripts containedthe 59 structure and 3, 6, 9, or 11 (G/U)AG repeats. As ex-pected, in the absence of TRAP we found that the cleavagepattern within the 59 stem-loop was essentially identical in allof the transcripts tested (Fig. 7). However, the cleavage patternof the various transcripts in the presence of bound TRAPdiffered considerably. When the transcript contained the firstthree (G/U)AG repeats (nucleotides 1 to 51), we observed areduction in cleavage following U11, A12, U23, and G24, aswell as increased cleavage following U25, A26, G31, C32, andU35 (Fig. 7). Note that the increase in cleavage following U25,A26, G31, and C32 was not as substantial as that observed forthe transcript that only contained the 59 stem-loop. The cleav-age pattern in the transcripts containing the first six (1 to 68)or nine (1 to 84) (G/U)AG repeats were similar to oneanother, although they differed from the other transcriptstested. RNase V1 cleavage was essentially absent followingU11, A12, U23, G24, A26, G31, and C32 (Fig. 7). Note thatthere was no increase in cleavage following U25, A26, G31,and C32 (Fig. 7). Remarkably, the RNase V1 cleavage patternin the transcript containing all 11 (G/U)AG repeats (1 to 111)was essentially identical to the pattern observed for thetranscript that only contained the 59 stem-loop. When takentogether, these results indicate that TRAP can interact with59 stem-loop in the absence of any downstream (G/U)AGrepeats and that the TRAP-59 stem-loop complex differs de-pending on the number of (G/U)AG repeats following the 59structure.

DISCUSSION

The transcription attenuation mechanism that controls ex-pression of the B. subtilis trpEDCFBA operon in response totryptophan relies on TRAP and three RNA secondary struc-tures. When TRAP binds to the 11 (G/U)AG repeats presentin the nascent trp leader transcript the antiterminator structurecannot form. Instead, an overlapping intrinsic terminator canform which results in transcription termination upstream of thetrp operon structural genes (Fig. 1). A recent genetic studydemonstrated that the 59 stem-loop also participates in thetranscription attenuation mechanism (28).

In the current study we examined the molecular basis of 59stem-loop function. We determined the secondary structure ofthe 59 stem-loop and found that it consists of a relativelyunstable 3-bp lower stem, a 5-by-2 asymmetric internal loop, a6-bp upper stem, and a hexaloop at the apex of the structure(Fig. 3 and 4). It is interesting to note that while both RNaseT1 and CMCT are single-stranded specific G probes, onlyCMCT detected G7 in the structure-mapping experiments.One possible explanation for this difference is that G7 partic-ipates in a non-Watson-Crick base-pairing interaction that pre-vents RNase T1 cleavage but leaves the N1 position availablefor CMCT modification. Our footprinting results suggest thatTRAP interacts with both sides of the asymmetric internalloop, the upper stem, the hexaloop, and the 39 side of the lowerstem (Fig. 5). It is interesting that the hexaloop contains aGAG sequence (nucleotides 18 to 20), while a single AAGsequence is present in the residues comprising the 39 side ofthe asymmetric loop and the 39 side of the lower stem (nucle-

FIG. 7. TRAP–59 stem-loop RNA footprint analysis with transcripts containing various numbers of (G/U)AG repeats. 59-end-labeled trp leader RNA containingthe 59 stem-loop and either 0, 3, 6, 9, or 11 (G/U)AG repeats was used in this analysis. trp leader RNA was treated with RNase V1 (1) in the presence (1) or absence(2) of tryptophan-activated TRAP. The positions of the nucleotides corresponding to the 59 stem-loop are indicated at the right. The relative positions of the 11(G/U)AG repeats, as well as G18, G24, and G31, are shown on the left. The lanes corresponding to partial alkaline hydrolysis (OH2) and partial RNase T1 digestion(T1) ladders generated from the transcript containing all 11 (G/U)AG repeats are indicated.


otides 29 to 31). The TRAP binding target in the trp leadercontains four UAG and seven GAG repeats between nucleo-tides 36 and 91 (Fig. 1) (8), while the TRAP binding site in theunlinked trpG transcript consists of one AAG, one UAG, andseven GAG repeats (15). Since it is known that 11 KKR motifson TRAP interact with GAG, UAG, and AAG repeats (2, 5,15, 31), it is possible that KKR motifs contribute to theTRAP–59 stem-loop complex by interacting with the GAGand/or AAG present within the 59 structure (Fig. 1). However,as pointed out in Results, substantial evidence suggests that theGAG sequence in the hexaloop interacts with a region ofTRAP that is distinct from the KKR motifs. If a KKR motifinteracts with the AAG sequence, then the spacing of fournucleotides between the AAG and the first UAG (nucleotides36 to 38) (Fig. 1) is suboptimal. The optimal spacing betweenrepeats is two nucleotides (7), although it was determined thatthree-nucleotide spacers are tolerated if present in the appro-priate context (9). Moreover, spacers of five and eight nucle-otides were identified in the trpG transcript (15); thus, it ispossible that a TRAP KKR motif interacts with this AAGsequence. This would bring the number of triplet repeats in theB. subtilis trp leader TRAP target to 12, the same numberidentified in the Bacillus stearothermophilus trp leader (12).Note that the UAG sequence (nucleotides 5 to 7) is unlikely tointeract with a TRAP KKR motif since deletion of the first fiveresidues had virtually no effect on trp operon expression (Table2).

Our results also indicate that TRAP interacts with the 59side of the internal loop and the upper stem (Fig. 5 and Table2). Moreover, we previously showed that substitution of G7with A resulted in a 59 stem-loop defect (28). Thus, it appearsthat TRAP interaction with the 59 side of the internal loopand/or non-Watson-Crick base pairing within this RNA seg-ment is crucial for 59 stem-loop function. Furthermore, wepreviously demonstrated that disruption of the upper stem bypoint mutations (C15G or G22C) had similar effects as delet-

ing the entire stem, while a C15G-G22C compensatory changeonly partially restored expression to wild type-like levels (28).This suggests that both the structure and the sequence of theupper stem are important for TRAP interaction.

While our footprinting and 59 stem-loop mutation studiesdemonstrated that TRAP interacts with the 59 structure andthat this interaction is required for proper regulation of the B.subtilis trp operon (Table 2), results from our boundary analysisindicate that TRAP–59 stem-loop interaction reduces the num-ber of downstream (G/U)AG repeats that are necessary fortight TRAP-trp leader RNA binding (Fig. 6). In addition, ourfootprinting results demonstrate that TRAP can interact withthe 59 stem-loop without any downstream repeats (Fig. 7).While the nature of the specific interactions are not well un-derstood, it is particularly striking that TRAP interaction withthe transcript containing only the 59 stem-loop resulted in a 59hairpin that was more highly structured. A qualitatively iden-tical result occurred when TRAP interacted with the transcriptcontaining the 59 structure and all 11 downstream (G/U)AGrepeats (Fig. 7). Interestingly, the TRAP-dependent RNase V1cleavage pattern that occurred in the transcripts containing the59 stem-loop and six or nine repeats were identical to eachother but clearly distinct from the cleavage pattern of the 0-and 11-repeat transcripts. Note that the TRAP-dependentcleavage pattern of the 59 stem-loop in the transcript that alsocontained three downstream (G/U)AG repeats is intermediatebetween the other two RNase V1 cleavage patterns. We believethat this static in vitro experiment captures the essence of thedynamic events taking place during transcription of the trpleader in vivo.

Our current model of the events taking place during tran-scription attenuation of the B. subtilis trp operon is shown inFig. 8. Soon after transcription initiates the 59 stem-loop forms(structure 1) (Fig. 8A). Tryptophan-activated TRAP subse-quently binds to structure 1, thereby promoting formation of amore highly structured 59 hairpin (structure 2). As transcrip-

FIG. 8. Transcription attenuation model of the B. subtilis trp operon. (A) Conditions of tryptophan excess. (B) Limiting tryptophan conditions. The 59 and 39 endsof the transcript are indicated. TRAP is represented by the gray doughnut structure. See the text for details.


tion proceeds, the KKR motifs on the TRAP perimeter inter-act with the (G/U)AG repeats one at a time as they becomeavailable, thereby wrapping the RNA around the periphery ofthe TRAP complex. Once all of the (G/U)AG repeats arebound, the geometry of this TRAP-trp leader RNA complex issuch that the trp leader transcript encircles the entire TRAP11-mer (2). Once this occurs the 59 stem-loop can dissociatefrom TRAP and retain the conformation of stem-loop struc-ture 2 or remain bound. The ability of the 59 stem-loop toremain bound is supported by the gel shift results, where weobserved increased TRAP affinity when the 59 stem-loop waspresent in a transcript that contained all 11 (G/U)AG repeats(Fig. 4), while dissociation is supported by the footprint anal-ysis (Fig. 7). As a consequence of TRAP binding, the antiter-minator structure cannot form, which promotes formation ofthe terminator structure and, hence, transcription termination.Since only a relatively short window of opportunity exists forTRAP to block antiterminator formation, it appears that thismultipartite binding mechanism increases the probability thatTRAP associates with the nascent trp leader transcript in timeto promote termination. When the concentration of trypto-phan is low, TRAP is not activated and does not bind to thenascent trp leader transcript. In this case, antiterminator for-mation prevents formation of the intrinsic terminator, result-ing in transcription of the entire operon (Fig. 8B). The trpoperon leader transcripts of Bacillus pumilus (18), Bacilluscaldotenax (31), and B. stearothermophilus (29) also contain 59stem-loops and multiple triplet repeats, as well as overlappingantiterminator and terminator structures. Thus, it appears thatall four organisms control expression of the trp operon byessentially identical transcription attenuation mechanisms.

ACKNOWLEDGMENTS

We thank Philip Bevilacqua, Craig Cameron, and Subita Suders-hana for discussions throughout the course of this study. We also thankPhilip Bevilacqua, Janell Schaak, and Charles Yanofsky for criticalreading of the manuscript.

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

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