©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structural Features of L-Tryptophan Required for Activation of TRAP, the trp RNA-binding Attenuation Protein of Bacillus subtilis(*)

Paul Babitzke (§) , Charles Yanofsky (¶)

From the (1) Department of Biological Sciences, Stanford University, Stanford, California 94305

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A filter binding assay was used to determine the structural features of L-tryptophan required for activation of TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis. We examined the ability of L-tryptophan and 26 of its analogs to activate TRAP. Our findings show that TRAP activation by L-tryptophan is highly cooperative. We also observed that TRAP activation is stereospecific; D-tryptophan did not activate. Our results further indicate that the -amino group and the carbonyl moiety of the -carboxyl group of the ligand are required for TRAP activation and that the heterocyclic amino nitrogen of L-tryptophan greatly enhances TRAP activation. We also found that changes at several positions of the indole ring of L-tryptophan resulted in reduced TRAP activation. In addition, indole and 5-methylindole were shown to be effective competitors of L-tryptophan activation of TRAP.


INTRODUCTION

The trp RNA-binding attenuation protein (TRAP)() of Bacillus subtilis is responsible for regulating expression of the tryptophan biosynthetic genes of this organism in response to changes in the intracellular level of L-tryptophan (Ref. 1 and references therein). Attenuation of the trpEDCFBA operon is mediated by the binding of TRAP, the product of mtrB, to the nascent trp leader transcript (2, 3, 4, 5, 6, 7) . In the presence of L-tryptophan, TRAP binds to a segment of the nascent trp transcript and prevents formation of or disrupts an RNA secondary structure, the antiterminator, thereby promoting formation of an overlapping rho-independent terminator that causes transcription termination at a site just preceding the trp structural genes (3) . In the absence of L-tryptophan, TRAP does not bind to the trp leader transcript (3, 6, 8) ; this allows the antiterminator to form, thereby preventing formation of the terminator and permitting transcriptional read-through into the trp structural genes (3) .

The one unlinked trp gene, trpG, is part of the folic acid biosynthetic operon (9) . TrpG is a bifunctional polypeptide involved in the biosynthesis of folic acid as well as L-tryptophan (10) . TRAP binds to a segment of the trpG transcript that includes the trpG ribosome-binding site (8, 9) , suggesting that TRAP binding regulates trpG translation by physically blocking ribosome access to the trpG ribosome-binding site.

Results from studies characterizing the RNA-binding targets of TRAP revealed that the TRAP-binding site in trp leader RNA consists of 11 equivalently spaced (G/U)AG repeats (7 GAG and 4 UAG), whereas the trpG-binding site contains 8 such repeats (7 GAG and 1 UAG), although the spacing between adjacent repeats is more variable in the trpG transcript (8) .

TRAP was recently crystallized in the presence of L-tryptophan and shown to be a complex composed of 11 identical 8328-Da subunits (11) . Electron microscopic studies indicate that the TRAP subunits are arranged in a single toroid ring.() In addition, several lines of evidence suggest that the TRAP subunit composition is identical in the presence or absence of L-tryptophan (3, 8) . Previous results demonstrated that the addition of L-tryptophan to TRAP altered the gel mobility of TRAP, suggesting that multiple L-tryptophan molecules can bind to TRAP (8) . These results are consistent with the results of equilibrium dialysis studies that showed that association of L-tryptophan and TRAP was cooperative, with one molecule of L-tryptophan bound per TRAP subunit (11).

In previous studies using an in vitro transcription attenuation assay, we observed that L-tryptophan, 5-fluoro-DL-tryptophan, and 5-methyl-DL-tryptophan could activate TRAP, whereas D-tryptophan, 1-methyl-L-tryptophan, 7-aza-DL-tryptophan, tryptamine, and indole-3-propionic acid could not (3) . In the present study we performed filter binding experiments with L-tryptophan and 26 of its analogs to determine the features of ligand required for TRAP activation.


MATERIALS AND METHODS

RNA Synthesis

Labeled transcripts were synthesized in vitro using T7 RNA polymerase according to the manufacturer's specifications (New England Biolabs Inc.). The labeling nucleotide was [5,6-H]UTP (approximately 8 Ci/mmol). Transcripts were separated from unincorporated nucleotides by using a Sephadex G-50 Quick Spin column (Boehringer Mannheim). The template used in the transcription reaction was pPB77 that had been linearized with BamHI; the resulting transcripts contained all 11 of the (G/U)AG trinucleotide repeats present in the wild type trp leader transcript (8) . The presence of a single RNA species of the appropriate size was confirmed by polyacrylamide gel electrophoresis.

Filter Binding Assays

TRAP was purified as described previously (3) . Filter binding assays were performed by a modification of an established procedure (8) . Standard filter binding reaction mixtures (0.1 ml) contained approximately 0.27 pmol (25 ng) of TRAP, 0.5 pmol of [H]RNA, 10 units of RNasin (Promega), 1 mML-tryptophan or one of its analogs, and 1 mM dithiothreitol in TKM buffer (40 mM Tris-HCl, pH 8.0, 250 mM KCl, 4 mM MgCl). TRAP dilution buffer was 50 mM Tris-HCl, pH 8.0, 50 mM KCl, and 15% glycerol (3) . Mixtures were incubated for 20 min at 37 °C and then filtered through BA85 nitrocellulose membranes (Schleicher & Schuell). The filters were washed twice with 0.1 ml of TKM buffer, dried, and counted in a liquid scintillation counter. Control experiments were performed in the absence of L-tryptophan or its analogs. Filter binding assays were performed in RNA excess. Modifications of the filter binding assay are described in the text, figure legends, and tables. L-Tryptophan and various tryptophan analogs were obtained from Sigma, Aldrich, or other commercial sources, except for 1-methyl-L-tryptophan, which was generously provided by Robert Phillips of the University of Georgia.


RESULTS AND DISCUSSION

TRAP Activation by L-Tryptophan Is Cooperative

Previous results demonstrated that L-tryptophan is responsible for TRAP activation (3, 6). In the presence of L-tryptophan, TRAP binds to a segment of the trp leader transcript containing 11 closely spaced (G/U)AG repeats and to a segment of the trpG message containing 8 such repeats (8) . Recent results have established that the TRAP multisubunit complex is composed of 11 identical subunits (11) . It was shown that one molecule of L-tryptophan can bind per TRAP subunit and that association of ligand with TRAP is cooperative (11) .

A filter binding assay that measures TRAP-trp leader RNA interaction (8) was used to analyze the effect on RNA binding as a function of increasing L-tryptophan concentration. The results of this analysis (Fig. 1) demonstrated that the ability of TRAP to bind RNA approached saturation at an L-tryptophan concentration of 10 µM with half-activation at approximately 5 µM. Under our conditions 2 µML-tryptophan was the lowest concentration that allowed TRAP to bind to the test transcript (Fig. 1). The sigmoidal shape of the activation curve indicates that L-tryptophan association with TRAP is highly cooperative. When taken together with previous findings demonstrating that one molecule of L-tryptophan can bind per TRAP subunit in a cooperative manner, these results suggest that one molecule of L-tryptophan/TRAP subunit may be required for full activation. The number of bound L-tryptophan molecules required for partial activation is not known (compare the activity at 2 and 10 µML-tryptophan in Fig. 1).


Figure 1: Cooperative activation of TRAP by L-tryptophan. TRAP was incubated with labeled RNA in the presence of increasing concentrations of L-tryptophan, and the mixtures were filtered. Background counts obtained in the absence of L-tryptophan were subtracted from each value. The number of counts/min (cpm) obtained following incubation with 1 mML-tryptophan was arbitrarily set at 100. Wild type trp leader RNA containing 11 (G/U)AG repeats was used in these analyses. See ``Materials and Methods'' for experimental details.



Cooperative activation suggests that association of the first molecule of L-tryptophan to unliganded TRAP might exert a conformational change in the TRAP complex that increases the likelihood that a second molecule of L-tryptophan will be bound. This process may be repeated until 11 L-tryptophan molecules are bound. Whatever the mechanism, however, the cooperative activation of TRAP by L-tryptophan differs from the mechanism of activation of Escherichia coli trp aporepressor (TrpR) by L-tryptophan. In the E. coli system, one molecule of L-tryptophan binds to each subunit of the TrpR repressor dimer noncooperatively (12, 13, 14, 15) .

Binding of TRAP to trp Leader RNA in the Presence of L-Tryptophan Analogs

Using an in vitro transcription attenuation assay, we previously observed that L-tryptophan, 5-fluoro-DL-tryptophan, and 5-methyl-DL-tryptophan could activate TRAP, whereas D-tryptophan, 1-methyl-DL-tryptophan, 7-aza-DL-tryptophan, tryptamine, and indole-3-propionic acid could not (3) . Subsequently a more sensitive filter binding assay was developed that allows us to examine TRAP activation by analogs directly (8) . To determine the features of L-tryptophan that are required for TRAP activation we used the filter binding assay to compare the ability of L-tryptophan and 26 of its analogs to activate TRAP (Fig. 2). Several analogs activated TRAP at a concentration of 1 mM; however, in no case was activation as effective as with L-tryptophan (). Analogs that resulted in appreciable TRAP activation at 1 mM were also tested at 50 and 5 µM. Five of the analogs were found to activate TRAP at 50 µM; however, none of these analogs activated appreciably at 5 µM ().


Figure 2: Structures of L-tryptophan analogs. Structures of L-tryptophan and 26 analogs were examined in these studies. The name of each compound is given below each structure.



TRAP Activation by L-Tryptophan Is Stereospecific

In a previous study using an in vitro transcription attenuation assay we found that D-tryptophan did not activate TRAP (3) . We tested D-tryptophan in the more sensitive filter binding assay to substantiate our previous result. D-Tryptophan did not activate TRAP at a concentration of 1 mM ( Fig. 2and ). This result, combined with the observations that 10 µML-tryptophan almost fully activated TRAP (Fig. 1) and that 5 µML-tryptophan resulted in 50% activation ( Fig. 1and ), demonstrates that TRAP activation is highly stereospecific, suggesting that positioning of the -carboxyl and amino groups of tryptophan is crucial for TRAP activation. This finding contrasts with results obtained for the E. coli trp repressor (TrpR), for which it was observed that, once bound, D-tryptophan is a more effective corepressor than L-tryptophan (16) .

The -Amino Group of L-Tryptophan Is Required for TRAP Activation

We previously reported that indole-3-propionic acid did not activate TRAP in the transcription attenuation assay (3) . This result suggested that the -amino group might be crucial for TRAP activation (Fig. 2). To examine this possibility more thoroughly, we used the filter binding assay to test analogs of L-tryptophan in which the -amino group of L-tryptophan was replaced by a hydrogen atom (indole-3-propionic acid) or a hydroxyl group (indole-3-lactic acid) or was methylated (N-methyl-L-tryptophan or L-abrine) (Fig. 2). Each of these changes eliminated TRAP activation (). These results suggest that the hydrogen donor capabilities and/or the positive charge of the -amino group is required for TRAP activation. Note that the simple presence of a positive charge is not sufficient for TRAP activation because both L-tryptophan and N-methyl-L-tryptophan (L-abrine) carry a positive charge (Fig. 2). These results are similar to findings with E. coli TrpR that indicated that the positive charge of the -amino group is required for corepressor activity. However, with TrpR L-abrine was found to be a better corepressor than L-tryptophan (16) .

The Carbonyl Moiety of the -Carboxyl Group Appears to Be Required for TRAP Activation

We previously observed, using a transcription attenuation system, that tryptamine did not activate TRAP (3). This result suggested that the -carboxyl group of L-tryptophan was involved in the activation of TRAP (Fig. 2). To substantiate this conclusion we used the filter binding assay to examine L-tryptophan analogs in which the -carboxyl group was replaced by a hydrogen (tryptamine) or a hydroxyl group (L-tryptophanol) or in which the negative charge was removed but the carbonyl moiety of the carboxyl group was retained (L-tryptophanamide and L-tryptophan methyl ester) (Fig. 2). The negative charge associated with the carboxyl group does not appear to be required for TRAP activation, because both L-tryptophan methyl ester and L-tryptophanamide activated TRAP at a concentration of 1 mM (). In fact, the methyl ester derivative resulted in substantial TRAP activation at a concentration of 50 µM (). However, removal of the carbonyl moiety and the negative charge (L-tryptophanol) or the entire carboxyl group (tryptamine) prevented TRAP activation (). These results suggest that the hydrogen acceptor capability of the carbonyl moiety is involved in TRAP activation and that the negative charge of the carboxyl group enhances but is not required for TRAP activation. These results are similar to findings with E. coli TrpR, for which it was shown that the -carboxyl group enhances but is not essential for corepressor activity (16) .

The Heterocyclic Amino Nitrogen of L-Tryptophan Greatly Enhances TRAP Activation

In studies with E. coli TrpR it was proposed that a substituent other than hydrogen at the N-1 position of L-tryptophan would sterically interfere with the protein-DNA contact surface and deprive the repressor-operator interaction of a stabilizing hydrogen bond (16) . We previously reported that 1-methyl-L-tryptophan did not activate TRAP (3) . This result seemed to indicate that the hydrogen donor capability of the indole ring's secondary amine was required for TRAP activation (Fig. 2). Using the more sensitive filter binding assay, we found that 1-methyl-L-tryptophan at a concentration of 1 mM resulted in low level TRAP activation (). This result indicates that the presence of the secondary amine, though not absolutely required, greatly enhances TRAP activation. Perhaps L-tryptophan donates the hydrogen atom at the N-1 position to a suitable hydrogen bond acceptor in TRAP.

TRAP Activation Is Reduced by Changes in the L-Tryptophan Benzene Ring

Several mtrB mutations have been isolated that are resistant to the L-tryptophan analogs 5-fluoro-L-tryptophan (4) and 5-methyl-L-tryptophan (17) . Because these analogs can activate TRAP (3) but are not suitable for protein synthesis (11) and because the drugs depress L-tryptophan synthesis, wild type cells presumably are inhibited. Thus, analog resistance arises by a loss of TRAP activity.

Results with the transcription attenuation assay indicated that the hydrogen atom at position 5 of the L-tryptophan benzene ring could be replaced by a fluoro (5-fluoro-L-tryptophan) or a methyl (5-methyl-L-tryptophan) group and still allow TRAP activation (3) . These and other analogs with benzene ring substitutions at positions 4, 5, or 6 were tested in the filter binding assay. Each of these analogs at a concentration of 1 mM activated TRAP (). The modifications included the replacement of a hydrogen with a methyl group at position 4 (4-methyl-L-tryptophan), a hydroxyl, fluoro, or methyl group at position 5 (5-hydroxy-L-tryptophan, 5-fluoro-L-tryptophan, or 5-methyl-L-tryptophan), or a fluoro or methyl group at position 6 (6-fluoro-L-tryptophan or 6-methyl-L-tryptophan). Of these, 5-hydroxy-L-tryptophan, 5-fluoro-L-tryptophan, and 6-fluoro-L-tryptophan activated TRAP at a concentration of 50 µM (). Notably, the level of TRAP activation by these analogs correlates with the physical size of the chemical substituent replacing the hydrogen; however, the relative polarity of the various substituents may effect TRAP activation ( Fig. 2and ).

Previous results suggested that substitution of a pyridine ring for the benzene ring of L-tryptophan (7-aza-L-tryptophan) eliminated TRAP activation (3). However, this compound gave significant TRAP activation at a concentration of 1 mM when it was tested in the more sensitive filter binding assay (). This result suggests that TRAP can tolerate a significant distortion of the heterocyclic ring of the indole moiety of L-tryptophan and still be activated. However, loss of the heterocyclic ring, as in L-phenylalanine and L-tyrosine, could not be tolerated ( Fig. 2and ).

Several other L-tryptophan analogs were also tested for the ability to activate TRAP in the filter binding assay. Of these, only substitution of the -hydrogen with a methyl group (-methyl-L-tryptophan) still permitted TRAP activation ( Fig. 2 and ). Taken together, the results of our TRAP activation studies indicate that TRAP can tolerate relatively modest changes in the structure of the indole ring of L-tryptophan; however, changes that disrupt the functional groups that may form hydrogen bonds with TRAP tend to have more serious consequences. Even a modest change such as methylation of the -amino group of L-tryptophan (N-methyl-L-tryptophan) completely abolished TRAP activation ( Fig. 2and ). In addition, changes that eliminate the carbonyl moiety of the -carboxyl group (L-tryptophanol and tryptamine) rendered the analog ineffective in TRAP activation, whereas analogs that retain the carbonyl moiety (L-tryptophan methyl ester and L-tryptophanamide) still can activate TRAP, albeit at substantially reduced levels in the case of L-tryptophanamide ( Fig. 2and ). Furthermore, disruption of the hydrogen bonding capability of the heterocyclic amino group of L-tryptophan (1-methyl-L-tryptophan) resulted in a dramatic decrease in its ability to activate TRAP ( Fig. 2and ).

Indole and 5-Methylindole Are Effective Competitors of L-Tryptophan

Previously, using the transcription attenuation assay, we found that D-tryptophan, 1-methyl-L-tryptophan, 7-aza-DL-tryptophan, tryptamine, and indole-3-propionic acid could not prevent TRAP activation when the analogs were present in a 50-fold molar excess over L-tryptophan (3) . We repeated and extended these L-tryptophan competition experiments using the filter binding assay with analogs that were unable to activate TRAP (). An L-tryptophan concentration of 5 µM was selected for analog competition analysis; at this concentration even slight competition will result in a substantial change in activation of TRAP (Fig. 1). Competition experiments were initially performed at an analog concentration of 1 mM (200-fold molar excess over L-tryptophan). Indole and 5-methylindole were very effective competitors; other analogs that were unable to activate TRAP showed only modest competition (data not presented). Competition by indole and 5-methylindole was examined at a range of concentrations, and both analogs were found to be much less effective at a 20-fold molar excess (100 µM) than at a 200-fold excess (1 mM) ( Fig. 2 and ). This requirement for excess competitor may reflect a weaker affinity for TRAP and/or the analogs may not induce cooperative binding. The fact that these analogs bind to TRAP and compete with L-tryptophan suggests that the indole ring is sufficient to confer attachment to the tryptophan-binding site(s) of TRAP. However, the indole moiety is insufficient to activate TRAP; as seen with other L-tryptophan analogs, the -carboxyl and -amino groups are required as well.

The results of these studies indicate that TRAP activation by L-tryptophan is highly cooperative and that TRAP activation is stereospecific. We determined that the -amino group and the carbonyl moiety of the -carboxyl group of the ligand are required for TRAP activation and that the secondary amine of the indole ring of L-tryptophan greatly enhances TRAP activation. We found that indole and 5-methylindole are effective competitors of L-tryptophan activation of TRAP, suggesting that the indole ring is recognized by the protein and that bound indole prevents L-tryptophan binding. However, indole and 5-methylindole did not activate TRAP.

  
Table: TRAP activation by various L-tryptophan analogs

Concentrations of compounds used are indicated. The number of counts/min (cpm) obtained with 1 mML-tryptophan was arbitrarily set at 100. Values are the averages of four experiments. See ``Materials and Methods'' for experimental details. ND, not determined.


  
Table: Inhibition of L-tryptophan activation of TRAP by indole and 5-methylindole

The number of counts/min (cpm) obtained with 5 µML-tryptophan alone was arbitrarily set at 100. The indole and 5-methylindole concentrations used are indicated. Values are the averages of four experiments. See ``Materials and Methods'' for experimental details.



FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM09738. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Postdoctoral Fellow of the National Institutes of Health. Permanent address: Dept. of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802.

Career Investigator of the American Heart Association. To whom correspondence should be addressed. Tel.: 415-725-1835; Fax: 415-725-8221.

The abbreviation used is: TRAP, trp RNA-binding attenuation protein.

P. Babitzke, D. G. Bear, and C. Yanofsky, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Bob Matthews and Peter Margolis for critical reading of the manuscript. We also thank Virginia Horn for technical assistance.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.