©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Kinetic and Thermodynamic Analysis of the Interaction between TRAP (trp RNA-binding Attenuation Protein) of Bacillus subtilis and trp Leader RNA (*)

(Received for publication, September 5, 1995; and in revised form, March 10, 1996)

Chris Baumann John Otridge (§) Paul Gollnick (¶)

From the Department of Biological Sciences, State University of New York at Buffalo, Buffalo, New York 14260

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In Bacillus subtilis, expression of the tryptophan biosynthetic genes is regulated in response to tryptophan by an RNA-binding protein called TRAP (trp RNA-binding attenuation protein). TRAP has been shown to contain 11 identical subunits arranged in a symmetrical ring. Kinetic and thermodynamic parameters of the interaction between tryptophan-activated TRAP and trp leader RNA were studied. Results from glycerol gradients and mobility shift gels indicate that two TRAP 11-mers bind to each trp leader RNA. A filter binding assay was used to determine an apparent binding constant of 8.0 ± 1.3 times 10^9M (K = 0.12 ± 0.02 nM) for TRAP and an RNA containing residues +36 to +92 of the trp leader RNA in 1 mML-tryptophan at 37 °C. The temperature dependence of K was somewhat unexpected demonstrating that the DeltaH of the interaction is highly unfavorable at +15.9 kcal mol. Therefore, the interaction is completely driven by a DeltaS of +97 cal mol K. The interaction between tryptophan-activated TRAP and trp leader RNA displayed broad salt and pH activity profiles. Finally, the rate of RNA dissociation from the RNAbulletTRAPbullettryptophan ternary complex was found to be very slow in high concentrations of tryptophan (>40 µM) but increased in lower tryptophan concentrations. This suggests that dissociation of tryptophan from the ternary complex is the rate-limiting step in RNA dissociation.


INTRODUCTION

In Bacillus subtilis, the genes for tryptophan biosynthesis are regulated in response to tryptophan by an RNA-binding protein called TRAP (trp RNA-binding attenuation protein; (1, 2, 3) ). TRAP regulates expression of the trp genes in three ways, all of which involve TRAP binding RNA in a tryptophan-dependent manner(3) . The trp operon (trpEDCFBA), which contains 6 of the 7 genes required for L-tryptophan biosynthesis, is regulated by transcription attenuation within a 204-nucleotide leader region preceding the first structural gene, trpE(1, 2) . In the presence of L-tryptophan, TRAP binds the nascent trp leader transcript (4) at a series of 11 G/UAG repeats between residues +36 and +91 (Fig. 1; (5, 6, 7) ). This binding prevents formation of an anti-terminator secondary structure which allows a transcription terminator to form and transcription halts in the leader region(1, 8) . When L-tryptophan is limiting, TRAP does not bind, the anti-terminator forms, and the operon is expressed.


Figure 1: Representation of residues +14 to +139 of trp leader RNA. The proposed terminator and anti-terminator structures are shown. Numbering is based on (1) and refers to the start of transcription. Residues in bold are the GAG and UAG repeats involved in TRAP binding. Asterisk indicates the residues proposed to be involved in the RNA-induced dimerization described under ``Discussion.''



TRAP also regulates translation of two trp genes, trpE (2, 9) and trpG(10) , apparently by two different mechanisms. Control of trpE translation is believed to be mediated by TRAP binding to the same series of G/UAG repeats in the leader segment of trp mRNAs that have escaped termination at the attenuator. This binding is thought to alter the RNA secondary structure so as to sequester the trpE ribosome binding site in a stem-loop structure, thus reducing translation initiation(2) .

trpG is the only tryptophan biosynthetic gene not located within the trp operon and is located within a folic acid biosynthetic operon(11) . Regulation of trpG translation occurs when TRAP binds a series of nine trinucleotide repeats which overlaps the putative ribosome binding site of trpG(5, 10) . Although the exact mechanism of control is not yet known, the data are consistent with a model involving direct competition between tryptophan-activated TRAP and ribosomes for trpG mRNA.

Recently, the three-dimensional structure of TRAP complexed with L-tryptophan has been solved by x-ray crystallography and refined to 1.8-Å resolution(6, 12) . TRAP is an endecamer, with the 11 identical subunits arranged in a symmetrical ring. The secondary structure of TRAP is formed entirely of beta-sheets, beta-turns, and random coils; no alpha-helices are present. TRAP is activated to bind trp leader RNA by binding 11 molecules of L-tryptophan in a highly cooperative manner(6) . The crystal structure reveals that each tryptophan molecule binds between two loops contributed by adjacent subunits; these loops contain residues 25-33 of one subunit and residues 49-52 of the other (6) .

Based on the structure of the TRAP endecamer and the multiple trinucleotide repeats in its RNA targets, we have proposed that upon binding, the RNA wraps around the protein such that each G/UAG repeat interacts with one subunit or a combination of two adjacent subunits of the TRAP 11-mer(6) . By wrapping around the protein, trp leader RNA would bind TRAP in a manner different from that observed in other characterized RNA-protein interactions(13) . Moreover, TRAP contains no sequences with similarity to other characterized RNA-binding motifs(14) .

Given the novel structure of TRAP and its RNA target, as well as the unusual manner by which tryptophan-activated TRAP is believed to bind trp leader RNA, it was of interest to examine the stoichiometry and thermodynamics of this interaction. Our results show that the activated form of TRAP binds trp leader RNA with a stoichiometry of two TRAP 11-mers to each RNA and an apparent binding constant of 8.0 ± 1.3 times 10^9M (K = 0.12 ± 0.02 nM). The free energy of interaction is driven by a large favorable change in entropy (DeltaS = +97 cal mol K) which is necessary to compensate for the substantial unfavorable change in enthalpy (DeltaH = +15.9 kcal mol). The lack of any significant salt dependence of the TRAP-RNA interaction (-log K/log [K] approx 0) indicates that electrostatic contacts do not play a major role in the stability of the complex and that the release of ions upon complex formation is not the major source of the favorable change in entropy. Previous work demonstrated that in the presence of excess tryptophan, the RNA in the tryptophanbulletTRAPbulletRNA ternary complex shows no significant dissociation after 30 min(5, 7) . Since TRAP functions as a regulatory protein by binding transcripts produced from the apparently unregulated trp promoter(1) , it seems unlikely that in vivo, the tryptophanbulletTRAPbulletRNA complex is this stable. To address this apparent paradox, we have studied the rate of RNA dissociation from the RNAbulletTRAPbullettryptophan ternary complex as a function of tryptophan concentration and have shown that the rate of dissociation of the ternary complex increases rapidly as the concentration of L-tryptophan is lowered.


MATERIALS AND METHODS

Plasmids

Plasmid pTZ18U14-92 contains residues +14 to +92 of the trp leader region (Fig. 1; (1) ) cloned downstream of the T7 promoter. It was constructed using polymerase chain reaction with pSPORTtrpL138 (4) as a template and primers trpL15 (5`-GCGAATTCAAGAGTGTGTATAAAGCAAT-3`) and trpL92 (5`-AGAAGCTTCTCAGCTCAACTAAACTCA-3`) complementary to residues +14 to +35 and to +92 to +73 of the trp leader DNA, respectively. The resulting polymerase chain reaction product was digested with EcoRI and HindIII and cloned into similarly cut pTZ18U (U. S. Biochemical Corp.). Plasmid pTZ18U36-92 was constructed in a similar manner except that primer trpL36 (5`-GCGAATTCTAGAATGAGTTGAGTTAGAG-3`) was used to create the 5` end of the construct starting at position +36 of the trp leader DNA.

RNA Synthesis and TRAP Purification

P-Labeled trp leader RNA was synthesized in vitro using T7 RNA polymerase and HindIII linearized templates as described previously(4) . For filter binding experiments, plasmid pTZ18U36-92 was used as the transcription template to generate RNA 36-92. Transcription reactions contained 500 pmol of unlabeled UTP and 100 µCi of [alpha-P]UTP (3000 Ci/mmol) yielding a final specific activity of 186 Ci/mmol UTP. Since RNA 36-92 contains 15 U's, the transcript had a final specific activity of 2790 Ci/mmol. Similarly, for stoichiometry experiments, plasmid pTZ18U14-92 or pTZ18U36-92 was transcribed in the presence 0.6 Ci/mmol [alpha-P]UTP to produce RNA 14-92 or RNA 36-92 with a specific activity of 12 Ci/mmol RNA 14-92 (20 U's/transcript) or 9 Ci/mmol RNA 36-92 (15 U's/transcript), respectively. All RNAs were purified on 10% polyacrylamide, 8 M urea gels and extracted by a crush and soak protocol(15) . TRAP was purified as described previously(12) .

In Vivo Labeling of TRAP

TRAP labeled with [S]methionine was prepared in vivo using a modification of the T7 RNA polymerase/promoter system(16) . Two 500-ml cultures of SG62052/pGP12 Escherichia coli cells (16) transformed with pTZ18UmtrB(4) , were grown in LB medium containing 100 µg/ml ampicillin and 50 µg/ml kanamycin at 30 °C. Upon reaching an A of 0.5, cells were harvested by centrifugation and washed once with 500 ml of M9 medium(17) . Each pellet was then resuspended in 1 liter of M9 medium (17) supplemented with 0.2% glucose, 20 µg/ml thiamine, and 18 amino acids (0.01%; minus cysteine and methionine). Cells were grown for 1 h at 30 °C and then heat shocked at 42 °C for 30 min. 200 µg/ml rifampicin and 10 mCi of [S]methionine (1175 Ci/mmol) were added and the cells were grown an additional hour at 37 °C before harvesting by centrifugation. Following two washes with M9 medium, S-labeled TRAP was purified by chromatography on phenyl-agarose as described previously(12) . The concentration of S-TRAP was determined spectrophotometrically to be 0.6 µg/µl based on the extinction coefficient of 1280 M cm at 280 nm, which was predicted by computer analysis (18) and confirmed by amino acid analysis(19) . The concentration of labeled TRAP was confirmed using two different colormetric assays (BCA, Pierce; Bradford, Bio-Rad) using purified, unlabeled TRAP as the standard. Based on these colormetric assays, the concentration of labeled TRAP was 0.58 ± 0.03 µg/µl and when averaged with the value obtained spectrophotometrically, a value of 0.59 µg/µl was used as the concentration of labeled protein. The specific activity of the S-labeled TRAP was determined by counting a known quantity of the labeled TRAP in scintillation mixture (Ecoscint, National Diagnostics).

Filter Binding Assays

For standard filter binding reactions, various concentrations of TRAP were equilibrated with 1 mML-tryptophan at room temperature (23 °C) in a total volume of 95 µl of FBB (250 mM potassium glutamate, 16 mM HEPES pH 8.0, 4 mM MgCl(2)). After 5 min, 5 µl containing 1 fmol of RNA 36-92 (1 times 10^4 dpm) and 10 µg of yeast tRNA were added and the reaction mixtures were incubated at 37 °C for an additional 15 min. 50-µl aliquots were then removed and filtered through a 0.45-µM cellulose nitrate membrane (Micro Filtration Systems) under vacuum. The filters were washed twice with 200 µl of FBB, dried, and counted in a Beckmann scintillation counter. Experiments at pH 5 and 6 were done in MES (^1)buffer, those at pH 8 were done in HEPES, and those at pH 10 were done in CAPS. The pH of all buffers was adjusted with either HCl or KOH.

For dissociation rate experiments, 0.8 nM TRAP was equilibrated with 40 µML-tryptophan for 5 min at room temperature in FBB. Then, 4 fmol of RNA 36-92 (4 times 10^5 dpm) were added to a total volume of 25 µl and incubated for 15 min at 37 °C. 975 µl of FBB containing various amounts of L-tryptophan were then added to yield final concentrations of L-tryptophan between 1 and 10.75 µM. 75-µl aliquots were removed at the indicated time points, filtered, washed, and counted as described above.

Velocity Sedimentation Centrifugation

Velocity sedimentation gradients to determine the stoichiometry of the TRAP-trp leader RNA complex contained 5-30% glycerol in GB (250 mM potassium glutamate, 4 mM Tris-HCl, pH 8, 12 mM HEPES pH 8, 4 mM MgCl(2), and 1 mML-tryptophan) in a final volume of 12 ml. 500-µl reactions containing GB, 20 µg of yeast tRNA, 1 pmol of radiolabeled RNA, and/or the appropriate concentration of S-TRAP were incubated on ice for 10 min. Following this incubation, 200-µl samples were applied to the surface of the gradient and the gradients were spun 48 h at 35,000 rpm in a SW41 rotor (Beckman) at 4 °C using slow acceleration, and deceleration without the brake. Gradients were collected from the bottom using a peristaltic pump at 13 ml/h. 360-µl fractions were collected and 200-µl aliquots were then counted in 10 ml of scintillation mixture (Ecoscint, National Diagnostics) and 1 ml of water. Two counting windows were used to measure the S and P in each aliquot. Window 1(0-688) contained 100% of the S and 33% of the P, whereas window 2(688-940) contained 67% of the P. The molar amounts of TRAP and RNA in each sample were calculated using the specific activities of TRAP and the RNA (see above).

Mobility Shift Gels

As an alternative method to separate the RNAbulletTRAP complex from the free forms of each, mobility shift gels were used. These gels were prepared and run as described previously (4) except N,N`-bis-acrylylcystamine (Bio-Rad) was substituted for bisacrylamide as the cross-linker. Samples containing 1 pmol of P-labeled RNA 36-92 and the indicated concentration of S-labeled TRAP were incubated on ice for 10 min in FBB with 1 mM tryptophan and then loaded on the gel. Following separation, the gel was exposed to a phosphorstorage screen and developed on a Molecular Dynamics PhosphorImager. Bands containing the TRAPbulletRNA complex were excised from the gel and dissolved by incubation in 300 µl of 1 M dithiothreitol for 1 h with gentle shaking. 200-µl aliquots were then mixed with 10 ml of scintillation fluid and 1 ml of H(2)O and counted as described above for the velocity gradient fractions.


RESULTS

Stoichiometry

Our model of the TRAP-trp leader RNA interaction, in which the bound RNA wraps around the protein (6) suggests a 1:1 TRAP-RNA stoichiometry in the complex. The stoichiometry of the TRAP-RNA complex was analyzed by using either glycerol gradients or mobility shift gels to separate the complex from the free RNA and TRAP. In both cases, we used S-labeled TRAP and P-labeled RNA and determined the molar ratio of TRAP and RNA in the complex based on the specific activity of each species.

The sedimentation profile of a glycerol gradient containing 5 pmol of S-labeled TRAP and 1 pmol of P-labeled RNA 14-92 is shown in Fig. 2. Nearly all of the RNA (Fig. 2, solid line, closed circles) was in fractions 14-19 which contained the TRAPbulletRNA complex, whereas TRAP (which was present in a 5-fold molar excess) was found in two peaks (Fig. 2, solid line, closed squares), one corresponding to the TRAPbulletRNA complex (fractions 14-19) and the second corresponding to the uncomplexed TRAP (fraction 20-25). Within the fractions containing the complex (Fig. 2, dashed line), the molar ratio of TRAP to RNA was 2.1 ± 0.2, n = 19. Similar results were obtained using 2.5-10 pmol of TRAP, as well as using an RNA containing the motif GAGUU repeated 11 times (data not shown). Control experiments showed that the free RNA and TRAP ran as single peaks within fractions 21-25 and 25-30, respectively, and were well separated from the TRAPbulletRNA complex (data not shown).


Figure 2: Glycerol gradient sedimentation of S-TRAPbulletP-trp leader RNA complex. Data shown were obtained with a trp leader RNA containing residues +14 to +92 relative to the transcription start site and the gradients contained 1 mML-tryptophan. Solid lines show the amount (femtomoles) of TRAP (closed squares) and RNA (closed circles) in each fraction. Dashed line shows the molar ratio of TRAP to RNA. y1 axis is the ratio of TRAP to RNA. y2 axis is the amount in femtomoles of TRAP and RNA. x axis is the fraction number with the top of the gradient being on the right.



We also used mobility shift gels to separate the TRAPbulletRNA complex from the free species of each (Fig. 3). Bands containing the complex were excised from the gel and the molar ratio of TRAP:RNA determined as above. In this case, RNA 36-92 was used and 18 bands were analyzed. The average ratio of TRAP to RNA in the complex was 2.8 ± 1.8. Although the variability seen in this case was higher than seen with the velocity gradients, the results are consistent with a dimer of 11-mers interacting with a single RNA. Taken together, these results indicate that under the conditions tested, two TRAP 11-mers form a complex with one molecule of trp leader RNA. Therefore, all binding data in this paper were analyzed using this stoichiometry.


Figure 3: Mobility shift gel using P-labeled RNA 36-92 and S-labeled TRAP. Each lane contained 1 pmol of RNA 36-92 and the indicated amount of TRAP. Experimental conditions were as described under ``Materials and Methods.'' Bands labeled as Complex correspond to the RNAbulletTRAP complex and were excised from the gel, dissolved in 1 M dithiothreitol, and the amount of RNA and TRAP in each band was determined as described in the text.



Equilibrium Binding Constants

As a tool to study the TRAP-RNA interaction, a filter binding assay was used with unlabeled TRAP and P-labeled RNA 36-92, which contains all 11 G/UAG repeats shown previously to be involved in TRAP binding (5, 6) and is not predicted to form any stable secondary structure (Fig. 1). To determine the incubation time needed to ensure that our binding assays were performed under equilibrium conditions, two control experiments were done. First, reaction mixtures containing RNA 36-92 and TRAP were incubated for either 15 min or 1 h and then filtered (Fig. 4, open circles, closed triangles, respectively). For either incubation time, a least-squares fit of the binding data to the equilibrium binding equation (20) displayed no significant differences. Second, equilibrium was established by dissociation. The RNAbulletTRAP complex was allowed to form at high concentrations of TRAP (0.44-200 nM) and RNA 36-92 (0.4 nM) such that nearly all of the RNA should be complexed with TRAP. After an initial 15-min incubation at 37 °C, the samples were diluted to the concentrations shown in Fig. 4, incubated for up to 6 h to re-establish equilibrium and then filtered (Fig. 4, closed stars). Again, the binding curve obtained was nearly identical to that observed when the mixture was allowed to incubate for 15 min (Fig. 4, compare closed triangles and open circles). Therefore, a 15-min incubation period is sufficient to allow our binding assay to achieve equilibration and this time was used for all subsequent experiments.


Figure 4: Equilibrium binding curve with trp leader RNA. The indicated concentration of TRAP was mixed with approx10 pMP-labeled trp leader RNA containing residues +36 to +92 (RNA 36-92) relative to the start of transcription(1) . The curves shown are the best fit of the filter binding data in the presence of 1 mML-tryptophan (open circles, closed triangles, and closed stars) and in the absence of L-tryptophan (closed squares) using a nonlinear least squares fitting algorithm. Control experiments, where the complex was allowed to form for an additional hour (closed triangles) and where equilibrium was reached by dissociation (closed stars) are shown and described in the text. Background retention in the absence of TRAP was subtracted from the binding data and was usually less than 2% of the input counts. The y axis is normalized to the total counts retained on the filter at saturation which, under standard buffer conditions, was greater than 65% of the input counts. Data are the average of at least four individual experiments.



Based on the best fit of the binding data, the apparent binding constant (K) for the TRAP-RNA interaction was determined to be 8.0 ± 1.3 times 10^9M (K(d) = 0.12 ± 0.02 nM). Based on the procedure of Witherell and Uhlenbeck (21) , we found that at least 65% of our TRAP preparation was active to bind RNA (data not shown). Therefore, the K reported above must be considered as a lower limit since less than 100% of the protein may be active. As shown previously(4, 5) , the interaction between TRAP and trp leader RNA is completely dependent on the presence of L-tryptophan since apo-TRAP, in the absence of tryptophan, showed no significant binding to trp leader RNA at any protein concentrations tested (Fig. 4, closed squares). Furthermore, a nonspecific RNA showed no binding to TRAP above background levels within the protein concentrations tested (data not shown).

Thermodynamics of the Interaction

To investigate the thermodynamic parameters that drive the interaction between TRAP and trp leader RNA, K values were determined for TRAP binding to trp leader RNA 36-92 at various temperatures. Surprisingly, K was found to increase with increasing temperature between 5 and 47 °C (Fig. 5). The van't Hoff plot of these data (Fig. 5) yields DeltaH = +15.9 kcal mol. This enthalpic contribution to the binding free energy is extremely unfavorable and is compensated by a large favorable entropic contribution, DeltaS = +97 cal mol K. Together, these values yield a DeltaG of -14.2 kcal mol and demonstrate that the binding of TRAP to trp leader RNA is entirely driven by changes in entropy.


Figure 5: Temperature dependence of K. Apparent binding constants for TRAP binding to RNA 36-92 obtained in FBB at the indicated temperatures (5-47 °C) and plotted as a van't Hoff plot. The slope of the best fit line is -8.0 and the y intercept = 48.7 yielding DeltaH = +15.9 kcal mol and DeltaS = +97 cal mol K based on the van't Hoff equation; ln K = (DeltaH/R)(1/Temp) + (DeltaS/R), where R = 1.987 times 10 kcal mol K.



Effect of Salt on Binding

To examine the role of electrostatic interactions in the TRAPbullettrp leader RNA complex, we determined K as a function of ionic strength using different concentrations of potassium glutamate with or without MgCl(2). In the presence of 4 mM MgCl(2) (Fig. 6, closed circles), binding was found to be insensitive to potassium glutamate concentrations between 100 and 700 mM (-log K/log [potassium glutamate] approx 0). In the absence of MgCl(2) (Fig. 6, open circles), K showed only a slight dependence on the concentration of potassium glutamate (-log K/log [potassium glutamate] approx 1). Similar results were obtained with NaCl or KCl (data not shown), indicating that the identity of the monovalent cation is not critical for stability of the complex. Furthermore, since neither KCl nor potassium glutamate had a significant effect on K, it appears that anion release is not a significant factor in complex formation. Therefore, it appears that few, if any electrostatic interactions play a major role in stabilizing the TRAPbullettrp leader RNA complex.


Figure 6: Ionic strength dependence of K. Apparent binding constants of the TRAP-trp leader RNA 36-92 interaction determined in FBB with the indicated potassium glutamate concentration (100-700 mM) plotted as a log-log plot. Solid circles, with 4 mM MgCl(2); open circles, without MgCl(2).



pH Effect on Binding

The role that titratable groups have on the interaction between TRAP and trp leader RNA was examined by determining the K for this interaction at pH values from 5 to 10. The K for TRAP binding to RNA 36-92 was found to be independent of pH within a range of pH 6 to 10 (Fig. 7). At pH 5, the K decreased 200-fold to 1 times 10^7M. The effects of pH on binding above pH 10 were not studied due to chemical degradation of the cellulose nitrate filters. This broad pH optimum suggests that groups titratable within this pH range are not significant in stabilizing the interaction.


Figure 7: Effect of pH on K. Apparent binding constants with RNA 36-92 obtained at 37 °C in FBB adjusted to the indicated pH. The following buffers were used: MES, pH 5 and 6; HEPES, pH 8; CAPS, pH 10. Data are the average of at least four experiments.



Dissociation of the TRAPbulletRNA Complex

Previous work (5) along with our own (data not shown) demonstrated that the TRAPbullettrp leader RNA complex is very stable in the presence of excess L-tryptophan showing no significant dissociation after 30 min. However, it seems unlikely that an RNA-binding regulatory protein such as TRAP would have such a slow off-rate in vivo since this would lead to the titration of TRAP, thereby leaving none available to bind new trp leader transcripts, resulting in the constitutive expression of the trp operon. To study this question, we examined dissociation of RNA from the tryptophanbulletTRAPbullettrp leader RNA ternary complex in the presence of various concentrations of L-tryptophan. We first allowed TRAP and trp leader RNA to form a complex in 25 µl of FBB with 40 µML-tryptophan, which was found to fully activate TRAP to bind trp leader RNA (data not shown; (22) ). The complex was then diluted 40-fold with 975 µl of FBB containing the appropriate concentration of tryptophan to yield the final tryptophan concentrations indicated in Fig. 8. To determine k, the binding data at each concentration of tryptophan were analyzed assuming a single exponential decay. We found that the dissociation rate for RNA was strongly dependent on the concentration of tryptophan. As the final concentration of tryptophan was lowered, k for the TRAPbulletRNA complex increased rapidly (Fig. 8). When the final concentration of tryptophan was 10.75 µM, k was 0.15 ± 0.07 min. As the final concentration of tryptophan was lowered to 5.9, 3.4, and 1 µM, k increased to 0.31 ± 0.06 min, 0.52 ± 0.07 min, and 1.8 ± 0.3 min, respectively. These results demonstrate that tryptophan can dissociate from the TRAPbulletRNAbulletL-tryptophan ternary complex and that this dissociation destabilizes the TRAP-RNA interaction. Therefore, dissociation of L-tryptophan from the ternary complex is the rate-limiting step for RNA dissociation.


Figure 8: Effect of L-tryptophan concentration of k. Dissociation experiments were performed by incubating TRAP (0.8 nM) and 0.16 nMP-labeled trp leader RNA 36-92 in FBB at 37 °C containing 40 µM tryptophan. At time 0, the complex was diluted 40-fold in FBB containing various amounts of tryptophan to yield the final concentration shown. Final concentration of tryptophan was: 10.75 µM (closed circles), 5.9 µM (closed squares), 3.4 µM (closed triangles), 1 µM (closed stars). Samples were filtered at various times and the counts retained were normalized to the counts at time 0, which were defined as 1. Data are the average of two independent experiments.




DISCUSSION

The x-ray crystal structure of TRAP complexed with L-tryptophan shows that TRAP is a homopolymer of 11 identical subunits arranged in a ring or ``doughnut'' structure(6) . It has also been shown that tryptophan-activated TRAP binds its RNA targets at a series of multiple G/UAG repeats(5, 6, 7) . From these findings, we have proposed that TRAP binds its RNA targets by the RNA forming a matching circle with the protein in which each trinucleotide repeat interacts with one subunit (or a combination of adjacent subunits)(6) , suggesting a 1:1 stoichiometry of TRAP and RNA in the complex. However, our results using glycerol gradients and mobility shift gels with S-labeled TRAP and P-labeled RNA suggest that two TRAP 11-mers bind each RNA. Interestingly, in crystals, TRAP was found to be a dimer of 11-mers lying ``face to face''(6, 12) . Furthermore, results from laser light scattering experiments (^2)indicate that while the majority of TRAP exists as an 11-mer in solution, a small percentage is found as a higher molecular mass complex of approximately 190,000 daltons, which is consistent with the predicted molecular mass of a dimer of 11-mers. Thus, it seems that the TRAP 11-mer may dimerize before or upon binding RNA. It remains to be seen if the TRAP dimer present in the crystals is the same as that which binds RNA.

Using a filter binding assay, an apparent binding constant of 8.0 ± 1.3 times 10^9M (K(d) = 0.12 ± 0.02 nM) was determined for the interaction between TRAP and trp leader RNA in FBB containing 1 mML-tryptophan at 37 °C. The data fit well to a simple bimolecular association curve indicating that only one complex is formed between L-tryptophan activated TRAP and this segment of trp leader RNA under these conditions. Analysis of this interaction by mobility shift assay also detected only a single complex (Fig. 3; (4) ). Similar results were obtained using an artificial TRAP binding site containing the motif GAGUU repeated 11 times which yielded a K of 8.6 times 10^9M (K(d) = 0.12 nM) (data not shown). It is interesting to note that the binding constant for the E. coli trp repressor for its DNA target is 0.2 nM(23) which is similar to the value we have observed for TRAP binding to its RNA target.

When several trp leader RNAs were used that contained 22 nucleotides 5` of the first UAG repeat (RNA 14-92 or RNA 14-115; see Fig. 1) a second inflection in the binding curve was observed (data not shown). This observation suggests that a second complex forms at high concentrations of TRAP with these RNAs. Since these 22 residues are not predicted to interact with TRAP upon RNA binding, we believe that they should be unconstrained in the complex. We propose that this second complex may be due to an RNA induced dimerization of the TRAPbulletRNA complexes caused by base pairing of the 5` end of the RNA in one complex with the 5` end of the RNA in the other (Fig. 1).

The interaction between tryptophan-activated TRAP and trp leader RNA differs in several ways from those observed for other RNA-protein interactions. For most RNA-protein interactions studied, DeltaH is approximately 0(13, 24, 25) , although R17 coat protein is a notable exception exhibiting a DeltaH = -19 kcal/mol(26) . However, the interaction between tryptophan-activated TRAP and trp leader RNA displays a very large unfavorable DeltaH of +15.9 kcal mol. In our model of the TRAP-RNA interaction we propose that the single-stranded trp leader RNA wraps around tryptophan-activated TRAP(6) . The observed DeltaH may represent the energy required to disrupt base stacking interactions within the RNA in order for it assume the proper conformation to bind TRAP.

Since the DeltaH of the interaction between tryptophan-activated TRAP and trp leader RNA is unfavorable, binding is driven entirely by a large favorable change in entropy. DeltaS is favorable for most RNA-protein interactions(13) , ranging from +13.1 cal mol K for TFIIIA (27) to +56 cal mol K for ermC` methyltransferase(28) . Again, R17 coat protein is an exception with an unfavorable DeltaS of -30 cal mol K(26) . To our knowledge, the interaction between TRAP and trp leader RNA has the highest DeltaS seen for an RNA-protein interaction, 97 cal mol K, which is needed to offset the highly unfavorable enthalpic contribution.

In general, a large favorable entropy change may be due to the release of either ions or water molecules upon complex formation(29) . To address the former possibility, the ionic strength dependence of TRAP binding trp leader RNA was investigated. In most RNA-protein interactions studied, the dependence of salt on binding is small with the -log K/log [M] being between 1 and 5(13) . For the TRAP-trp leader RNA interaction, K is not dependent on the concentration of potassium glutamate in the presence of MgCl(2) with -log K/log [K] approx 0 (Fig. 6, closed circles). Without MgCl(2), there is only a slight dependence of the K on the concentration of potassium glutamate equivalent to approx1 ionic interaction per complex. If ionic interactions were involved in the binding, there would likely be at least 11 ionic contacts being made. This assumption is based on our model of the TRAP-RNA interaction where each of the 11 subunits would contact one of the 11 trinucleotide repeats(6) . Therefore, if ionic contacts were significantly involved in stabilizing the complex, we would predict that the binding would have a much greater ionic strength dependence than observed. Our findings do not completely rule out the possibility that ion release is involved in the favorable change in entropy but it does make it likely that the release of water molecules is a major driving force in the binding.

Previous work has suggested that TRAP binds single stranded RNA(2, 5) . The lack of MgCl(2) dependence on complex formation is consistent with this hypothesis since Mg is often involved in stabilizing RNA secondary and tertiary structures(30) . We have observed that RNA 36-92, RNA 14-92, and an artificial TRAP binding site containing the motif GAGUU repeated 11 times, none of which are predicted to form any stable secondary structures, all bind TRAP approximately 3-fold better than an RNA containing residues +14 to +115 (data not shown), which is capable of forming the anti-terminator structure (Fig. 1). Furthermore, it has been shown that when the TRAP binding site is placed in a perfect stem-loop, this RNA does not bind TRAP. (^3)Taken together, these observations support the model that TRAP binds trp leader RNA in a single stranded form.

We have shown that the TRAPbulletRNA complex dissociates rapidly when diluted into lower concentrations of tryptophan. Along these lines, the concentration of tryptophan where RNA dissociation is observed was found to be subsaturating for the TRAP-tryptophan interaction under similar conditions (data not shown). This observation suggests that tryptophan is able to dissociate from the RNAbulletTRAPbullettryptophan ternary complex resulting in the destabilization of the remaining TRAPbulletRNA complex. If the concentration of tryptophan is too high, such that TRAP remains completely bound by tryptophan, the ternary complex dissociate very slowly. Therefore, it is reasonable to propose that in B. subtilis, the in vivo concentration of free tryptophan should not be allowed to rise to a concentration where TRAP will not dissociate from trp leader RNA since this would lead to the deregulation of the trp operon due to the titration of TRAP. This contrasts the situation in E. coli, where at high concentrations of free tryptophan, the trp repressor simply remains bound to its DNA binding site and prevents transcription initiation from the trp promoter. Moreover, excess free tryptophan in E. coli can also be used as a source of carbon and nitrogen due to the products of the tryptophanase operon(31) , which is lacking in B. subtilis.

In the crystal structure, 11 molecules of L-tryptophan bind TRAP in deep clefts between adjacent subunits and are completely buried within the protein with no exposure to the solvent. This binding is proposed to activate TRAP to bind RNA by inducing conformational changes in two loops formed by residues 25-33 on one subunit and 49-52 on the other(6) . We have proposed that the RNA binds to TRAP at regions affected by tryptophan binding and these two loops seemed likely candidates for the RNA binding domain in TRAP(6) . However, if the RNA bound to these loops, it is unlikely that L-tryptophan would still be able to freely dissociate from the ternary complex as we have seen. This observation suggests that although these loops are important for tryptophan binding and likely play a role in the activation of TRAP, the RNA interacts with another region of the protein. Recent results from our laboratory using site-directed mutagenesis support this hypothesis, indicating that the RNA binding site of TRAP involves residues on beta-sheets located near the outer edge of the TRAP doughnut. (^4)

In this report, we have characterized several aspects of the interaction between tryptophan-activated TRAP and trp leader RNA. Binding is driven completely by changes in entropy yet is not ionic strength dependent, suggesting that the interaction may be driven by the release of ordered water molecules. The lack of ionic strength dependence also implies that the TRAPbulletRNA complex is mediated mostly by hydrophobic interactions and hydrogen bonds rather than ionic interactions. Finally, the dissociation of the complex suggests that L-tryptophan may play a role in the regulation of the trp operon in vivo by not only controlling the association of TRAP and RNA but by also controlling the rate of dissociation of the TRAPbulletRNA complex.


FOOTNOTES

*
This work was supported by National Science Grant MCB-9118654. 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.

§
Current address: National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Health, Genetics and Biochemistry Branch, 10 Center Dr., MSC1766, Bethesda, MD 20892-1766.

A Pew Scholar in the Biomedical Sciences. To whom correspondence should be addressed. Tel.: 716-645-2887; Fax: 716-645-2975.

(^1)
The abbreviations used are: MES, 4-morpholineethanesulfonic acid; CAPS, 3-(cyclohexylamino)propanesulfonic acid.

(^2)
Wyatt Technologies and P. Gollnick, unpublished results.

(^3)
P. Babitzke, personal communication.

(^4)
M. Yang, B. Fernandez, and P. Gollnick, unpublished results.


ACKNOWLEDGEMENTS

We thank Shannon Hilchey, Barry Hurlburt, Gerald Koudelka, and Charles Yanofsky for critical reading of the manuscript and Xiaoping Chen for technical assistance. We also thank Paul Babitzke for sharing results prior to publication and Edward Brody and Shasi Harvey for help with the glycerol gradients.


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