THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 277, No. 14, Issue of April 5, pp. 11838 –11844, 2002 Printed in U.S.A.

Creating Hetero-11-mers Composed of Wild-type and Mutant Subunits to Study RNA Binding to TRAP* Received for publication, November 13, 2001, and in revised form, January 9, 2002 Published, JBC Papers in Press, January 22, 2002, DOI 10.1074/jbc.M110860200

Pan T. X. Li‡§, David J. Scott¶储, and Paul Gollnick‡** From the ‡Department of Biological Sciences, State University of New York, Buffalo, New York 14260 and the ¶York Structural Biology Laboratory, Department of Chemistry, University of York, York Y010 5DD, United Kingdom

In Bacillus subtilis, expression of the tryptophan biosynthetic (trp) genes is negatively regulated by the trp RNA-binding attenuation protein TRAP. In the presence of excess tryptophan, TRAP binds to several RNA targets that each contain between 9 and 11 (G/U)AG repeats usually separated by 2 or 3 nonconserved nucleotides (1– 4). TRAP regulates transcription of the trpEDCFBA operon through an attenuation mechanism (5– 8). In addition, TRAP also regulates translation of at least three genes including trpG, yhaG, and trpE. TRAP binding to the leader region of trp operon read-through transcripts alters the RNA structure so as to sequester the trpE Shine-Dalgarno sequence (9, 10). For trpG TRAP competes with ribosomes for binding to the mRNA (11, 12); this mechanism also appears to regulate translation of yhaG (13). Structures of TRAP (1, 14) and of TRAP complexed with an

* This work was supported by Grant GM62750 from the National Institutes of Health and Grant MCB 9982652 from the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Present address: Dept. of Chemistry, University of California, Berkeley, CA 94720. 储 Present address: Dept. of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom. ** To whom correspondence should be addressed.

RNA containing 11 GAG repeats (15) have been determined. TRAP consists of 11 identical subunits (75 amino acids each) arranged in a symmetric ring termed the ␤-wheel (Fig. 1) (1). The ␤-wheel consists of 11 seven-stranded antiparallel ␤-sheets, each made of four strands from one subunit and three strands from an adjacent subunit. The oligomeric structure is maintained by hydrophobic interactions and hydrogen bonds between ␤-strands. There are 11 tryptophan-binding sites on TRAP, each formed at the interface of adjacent subunits. Single-stranded RNA containing GAG or UAG repeats binds to TRAP by wrapping around the outer perimeter of the ␤-wheel forming specific hydrogen bonds between the bases of the RNA and amino acids of the protein (15).1 Based on studies of nucleoside analog substitution in the RNA, Elliott et al. (16) proposed a two-step model for RNA binding to TRAP in which binding initiates with a single RNA triplet repeat forming an initiation complex, followed by cooperative binding of the remaining repeats. Here we report that the native TRAP 11-mer can be reversibly denatured into unfolded monomers by guanidine hydrochloride (GdnHCl).2 This finding allowed us to develop a method to mix different types of TRAP subunits to generate heteromeric TRAP 11-mers. Studies of RNA binding to these heteromeric TRAP 11-mers further support and extend the two-step model by showing that the initiation complex requires at least one fully active subunit in the protein combined with a fully functional repeat in the RNA. EXPERIMENTAL PROCEDURES

Materials—GdnHCl (ultra pure, electrophoresis grade) was purchased from Angus Buffers and Biochemicals (Niagara Falls, NY). An 8.0 M stock solution was made and filtered through a 0.45-␮m membrane filter (Nalgene). L-Tryptophan was obtained from Fisher. RNA Synthesis and TRAP Purification—(GAGUU)11, an RNA containing 11 tandem repeats of the sequence GAGUU, was transcribed in vitro using T7 RNA polymerase and [␣-32P]UTP (PerkinElmer Life Sciences) as described by Baumann et al. (17). Wild-type and mutant TRAP proteins were expressed in Escherichia coli and purified by immunoaffinity chromatography as described previously (18). All mutant proteins used in this paper have been described previously (19). Protein concentrations were determined by UV absorbance at 280 nm using an extinction coefficient of 1280 M⫺1 cm⫺1 and confirmed by the BCA protein assay (Pierce) and SDS gel electrophoresis with comparisons to TRAP standards of known concentration. Tryptophan and RNA Binding—Tryptophan binding to TRAP was analyzed by equilibrium dialysis using [14C]L-tryptophan (PerkinElmer Life Sciences) as described previously (1). RNA binding affinity in the presence of excess L-tryptophan (1 mM) was measured using a modification of the filter binding assay described by Baumann et al. (17). Denaturing and Renaturing TRAP—TRAP was denatured in ⱖ4.0 M GdnHCl for at least 30 min at room temperature. Denatured TRAP was renatured by dialysis against 50 mM phosphate buffer (pH 8.0) overnight. When comparing the denaturation and renaturation pathways,

1 2

11838

A. Antson and P. Gollnick, unpublished results. The abbreviation used is: GdnHCl, guanidine hydrochloride. This paper is available on line at http://www.jbc.org

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TRAP (trp RNA-binding attenuation protein) is an RNA-binding protein that regulates expression of the tryptophan biosynthetic genes in Bacillus subtilis by binding to RNA targets that contain multiple GAG and UAG repeats. TRAP is composed of 11 identical subunits arranged symmetrically in a ring. The secondary structure of the protein consists entirely of antiparallel ␤-sheets, ␤-turns, and loops. We show here that the TRAP 11-mer can be reversibly denatured into unfolded monomers by guanidine hydrochloride. Removing the denaturant allows the protein to spontaneously renature into fully functional 11-mers. Based on this finding, we developed a subunit mixing method to hybridize wild-type and mutant subunits into heteromeric 11mers by denaturation followed by subunit mixing renaturation. This method allows the study of subunit cooperativity in protein-ligand interaction such as RNA binding. Our data further support and extend the previously proposed two-step model for RNA binding to TRAP by showing that the initiation of binding requires at least one fully active subunit in the protein combined with one fully functional repeat in the RNA. The initiation complex tethers the RNA on the protein, thus allowing cooperative interaction with the remainder of the repeats.

TRAP-RNA Interaction by Subunit Mixing

11839

we started with the same concentration of TRAP in either 50 mM sodium phosphate buffer (pH 8.0) or in 6.0 M GdnHCl. The samples were diluted with either 8.0 M GdnHCl or phosphate buffer, respectively, to the desired final concentration of GdnHCl and incubated at least 2 h. Subunit Mixing—Various ratios of mutant and wild-type TRAP proteins were mixed together in GdnHCl for at least 2 h. The mixtures were then dialyzed against 50 mM sodium phosphate buffer (pH 8.0) overnight. The recovered samples were electrophoresed on 9% native polyacrylamide gels using a Bio-Rad Mini-Protean II system at 30 mA for 4.5 h. The gels were stained with Coomassie Brilliant Blue and photographed with a Kodak DC290 digital camera. The gel images were analyzed by GeneImager software (Scanalytics Inc). Circular Dichroism Measurements—CD spectra were obtained on a JASCO model J-700 spectropolarimeter. Samples containing 12 ␮M TRAP 11-mer were scanned in 1-mm quartz cuvettes at room temperature. Spectra of GdnHCl at the appropriate concentrations were subtracted as background. Analytical Ultracentrifugation—Analytical ultracentrifugation experiments were carried out using a Beckman XL-A analytical ultracentrifuge using an AnTi-60 rotor. The samples were loaded at set GdnHCl concentrations into cells with six channel centerpieces. Equilibrium was attained at 10,000, 15,000, and 20,000 rpm at 20 °C. Buffer density was calculated using the method of Laue et al. (20); contribution to the partial specific volume caused by the presence of high concentrations of GdnHCl was accounted for using the method of Arakawa and Timasheff 0 (21). The whole cell molecular mass Mw, app was determined using the program MSTAR (22). The M* function is an operational point average molecular mass given by the following equation. r

冕

C共r兲 ⫺ Ca ⫽ kCa共r2 ⫺ a2兲 ⫹ 2k r关C共r兲 ⫺ Ca兴dr M*共r兲

(Eq. 1)

a where C(r) is the concentration at radius r, Ca is the concentration at the radial position of the meniscus, a, M* is the operational point average molecular mass, k ⫽ (1 ⫺ ៮v␳)␻2/2RT, where ៮v is the partial specific volume, ␳ is the density, ␻ is the angular velocity, R is the universal gas constant, and T is the thermodynamic temperature. Size Exclusion Chromatography—Size exclusion chromatography was performed using a Biosep SEC-S2000 size exclusion column (7.8 ⫻ 300 mm; Phenomenex) and a PerkinElmer Life Sciences series 8800 HPLC system. Samples containing 10 –20 ␮g of TRAP in 50 mM sodium phosphate (pH 8.0) with various concentrations of GdnHCl were eluted

FIG. 2. Denaturing the secondary structure of TRAP with GdnHCl. A, circular dichroism spectra of 12 ␮M TRAP 11-mers in 50 mM Na2HPO4 (pH 8.0) (f); in 6.0 M GdnHCl (E); and after incubating in 6.0 M GdnHCl for 30 min followed by dialysis into Na2HPO4 (pH 8.0) (〫). The spectrum below 210 nm was not shown for TRAP in 6.0 M GdnHCl because of high background of GdnHCl in this range. Each curve represents the average of 10 scans with background subtracted. B, unfolding and refolding TRAP as monitored by CD at 215 nm at 25 °C. For unfolding (f), GdnHCl was added to TRAP in Na2HPO4 (pH 8.0) to give the desired final concentration of denaturant. For refolding (E), TRAP was incubated in 6.0 M GdnHCl for 30 min and then diluted with Na2HPO4 (pH 8.0) to give the desired final concentration of GdnHCl. The final concentration of TRAP 11-mer is 12 ␮M for all samples. Background from buffer with at the same concentration of GdnHCl was subtracted for each sample. by the same buffer at a flow rate of 0.5 ml min⫺1. The detection wavelength was 215 nm. RESULTS

GdnHCl Reversibly Denatures TRAP 11-mer into Unfolded Monomers—Previous studies have shown that the TRAP 11mer is very stable and is resistant to denaturation by heat (23) or by urea or SDS.3 Here, we show that TRAP can be reversibly denatured into unfolded monomers by guanidine hydrochloride. In native conditions, the CD spectrum of TRAP is typical of a ␤-sheet dominant protein (23, 24), showing a positive peak at 205 nm and two negative peaks at 190 and 217 nm, respectively (Fig. 2A). This spectrum is consistent with the crystal structure of TRAP, which shows that the secondary structure is composed of ␤-strands connected by ␤-turns and loops (1) (Fig. 1). In contrast, the CD spectrum of TRAP in 6.0 M GdnHCl (Fig. 2A) is indicative of a random coil, because it is nearly identical to that of poly-L-glutamine in random coil conformation (25). We were unable to obtain a reproducible CD signal for TRAP below 210 nm in the presence of 6.0 M GdnHCl because of high 3

P. Gollnick, unpublished observations.

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FIG. 1. The structure of TRAP. Ribbon diagram of the TRAP 11mer with one subunit shown darkly shaded. The bound L-tryptophan molecules are shown as van der Waal’s spheres.

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TRAP-RNA Interaction by Subunit Mixing

FIG. 3. Subunit dissociation of the TRAP 11-mer. A, monitoring dissociation of TRAP by analytical ultracentrifugation. The apparent molecular mass of TRAP in various concentrations of GdnHCl was measured by analytical ultracentrifugation. The whole cell molecular mass was determined after reaching equilibrium at 20 °C. Each point represents the average of three independent experiments with standard deviation less than 10% of the mean. B, monitoring dissociation of TRAP by size exclusion chromatography. Samples containing 10 –20 ␮g of TRAP in various concentrations of GdnHCl in Na2HPO4 (pH 8.0) were chromatographed on a Biosep SEC-S2000 (Phenomenex) size exclusion column at 0.5 ml min⫺1. Detection was at 215 nm.

We next examined the effects of GdnHCl on TRAP function. Under native conditions, TRAP binds 11 L-tryptophan molecules cooperatively with an affinity (S0.5) of 5–10 ␮M and a Hill coefficient (n) of 1.5–2.0 (1, 19, 28) (Fig. 4A). In the presence of ⬎2.0 M GdnHCl, we were unable to measure any tryptophan binding to TRAP. However, after dialyzing samples treated with 2.0 – 6.0 M GdnHCl, the proteins showed similar tryptophan binding properties as the native protein. Similar results were also obtained when we examined the RNA binding properties of TRAP. TRAP binds to (GAGUU)11, an RNA containing 11 tandem repeats of the sequence GAGUU, with a Kd of 1 nM (4) (Fig. 4B). Again in the presence of ⬎2.0 M GdnHCl TRAP shows almost no detectable RNA binding activity, whereas

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background from the denaturant. The molar ellipticities at 217 nm for TRAP, 16.8 deg cm2 dmol⫺1 (native) and 5.8 deg cm2 dmol⫺1 (6.0 M GdnHCl) are consistent with reported values for ␤-sheet and for random coil proteins, respectively (24). Therefore, these results indicate that TRAP is unfolded into random coils in 6.0 M GdnHCl. When the GdnHCl is removed by dialyzing the samples in sodium phosphate buffer (50 mM, pH 8.0), the CD spectrum of the protein is, within experimental error, identical to that of native TRAP (Fig. 2A), suggesting that the secondary structure of the protein has been renatured. To follow unfolding/refolding of the secondary structure of TRAP by GdnHCl, we collected CD spectra at various concentrations of denaturant. The samples were prepared either by adding various amounts of 8.0 M GdnHCl to TRAP in phosphate buffer (denaturation) or by adding buffer to TRAP samples after incubation in 6.0 M GdnHCl (renaturation). The spectra obtained by both approaches were nearly identical at the same final concentration of GdnHCl, indicating that the unfolding of TRAP by GdnHCl is reversible. Fig. 2B shows the data for both renaturation and denaturation followed at 215 nm, which is the wavelength where the largest difference between the spectra in Fig. 2A was observed. The largest changes in ellipticity occur between 0 and 2.5 M GdnHCl, indicating significant loss of the secondary structure of TRAP over this range of denaturant. The spectra indicate that at ⱖ2.5 M GdnHCl the protein exists mainly as a random coil. To examine the oligomeric state of the protein during denaturation, we performed analytical ultracentrifugation of TRAP in various concentrations of GdnHCl. The predicted molecular mass of a TRAP subunit is 8,325 Da, which has been confirmed by mass spectroscopy (R. Philips, University of Georgia); thus the molecular mass of the TRAP 11-mer is 91.6 kDa. For each sedimentation equilibrium profile at a given concentration of GdnHCl, we obtained the whole cell molecular mass average, M0, app, which is plotted versus [GdnHCl] in Fig. 3A. M0, app falls from a value (93 kDa) closely corresponding to the 11-mer in 0.0 M GdnHCl to that corresponding to the monomer (8 –9 kDa) in ⱖ3.0 M GdnHCl. These experiments further show that TRAP is an 11-mer in less than ⱕ0.5 M GdnHCl and monomers at ⱖ 3.0 M GdnHCl. Similarly, the fluorescence anisotropy of TRAP, which measures the rotational diffusion of the molecule in solution, drops from 0.12 in 50 mM phosphate buffer (pH 8.0) to 0.02 in ⱖ2.5 M GdnHCl (data not shown). The former value is close to that of bovine serum albumin (molecular mass, 66 kDa) or aldolase (molecular mass, 158 kDa) in phosphate buffer, and the latter value is similar to that of free tyrosine or tryptophan in either phosphate buffer or 6.0 M GdnHCl. Together, these experiments show that TRAP exists as an 11-mer in less than 0.5 M GdnHCl and monomers at ⱖ3.0 M GdnHCl. We also obtained similar results by size exclusion chromatography (Fig. 3B). In the absence of GdnHCl (50 mM phosphate buffer, pH 8.0), TRAP eluted as a single peak at 8.5 min, which based on molecular mass standards (Amersham Biosciences, Inc.) corresponds to a molecular mass of 60 –70 kDa. This result is consistent with previously published observations (18, 26) showing that size exclusion chromatography underestimates the molecular mass of native TRAP (91.6 kDa), which is probably due to the circular shape of this protein. At ⱖ3.0 M GdnHCl, the protein elutes as a single peak at 9.7 min correlating to monomers. At intermediate concentrations of GdnHCl between 0.5 and 3.0 M, we observe only the two peaks corresponding to the 11-mer and the unfolded protein (Fig. 3B). These findings support a two-state oligomerization model in which TRAP exists only as monomers or as 11-mer under the experimental conditions (27).

TRAP-RNA Interaction by Subunit Mixing

after renaturation by dialysis, the GdnHCl-treated proteins bind RNA with similar properties as native TRAP. Subunit Mixing to Create Heteromeric TRAP 11-mers—Together the studies described above demonstrate that we can use GdnHCl to denature TRAP into unfolded monomers and then reform fully functional 11-mers by simply removing the denaturant. These observations suggested that it could be possible to mix denatured subunits from two different types of TRAP proteins (i.e. mutant and wild type) and then reassemble heteromeric 11-mers composed of both types of subunits. If the two types of subunits (A and B) assemble randomly, then mixing subunits into 11-mers would result in 12 possible combinatory species: A11, A10B, A9B2 . . . AB10, and B11, and the relative abundance of each type of oligomer in the mixture would follow the binomial distribution. It is also possible that the subunits could assemble nonrandomly, with the most extreme case being total exclusion of hetero-interactions, in which case only the two original homo-11-mers (A11 and B11) would form upon renaturation. To follow subunit mixing, we took advantage of TRAP mutants in which the charge of the substituted residue is changed.

FIG. 5. Mixing wild-type and K71A mutant TRAP subunits to create heteromeric TRAP 11-mers. A, native polyacrylamide gel electrophoresis of WT-K71A hetero-11-mers. Wild-type and K71A (Lys71 substituted with Ala) TRAP were denatured in 6.0 M GdnHCl for 30 min, mixed in various ratios, dialyzed into Na2HPO4 (pH 8.0), and then electrophoresed on a 9% native polyacrylamide gel. The gel was stained with Coomassie Brilliant Blue. B, histogram of the observed and predicted intensities of the bands in the gel shown in A. The intensity of each band in lanes 3– 8 of the gel shown in A was determined using GeneImage software and is plotted as gray bars. The predicted intensities based on binomial distribution is presented in black bars for comparison.

Such mutants have altered mobility on native polyacrylamide gels as compared with wild-type TRAP. Examples of such mutant proteins that we have studied include K37A, K56A, R58A, and K71A in which either lysine or arginine residues on the surface of the protein are replaced by alanine at the position indicated. Previous studies showed that these mutant proteins exist as 11-mers (19). These substitutions result in negligible changes (⬍1%) in molecular mass but are predicted to change the isoelectric point of the mutant TRAP 11-mers by about 0.5 pH units, which we confirmed by isoelectric focusing gel electrophoresis (data not shown). Hence, the mobility differences of these proteins on native gels are most likely due to the difference in net charge of the proteins at pH 8.3. Fig. 5A shows an example of subunit mixing displayed on a native polyacrylamide gel, where wild-type and K71A mutant TRAP proteins were denatured in 5.0 M GdnHCl, mixed in different ratios, and then dialyzed against 50 mM phosphate buffer (pH 8.0) overnight. The recovered TRAP proteins have CD spectra very similar to those of wild-type TRAP and were

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FIG. 4. RNA binding and tryptophan binding properties of renatured TRAP. A, equilibrium dialysis measurements of 14C-Ltryptophan binding to native TRAP (f); TRAP in 3.0 M GdnHCl (●); and renatured TRAP (Œ). The curves were fit into the Hill equation. Renatured TRAP was treated with 6.0 M GdnHCl for 30 min and then dialyzed into Na2HPO4 (pH 8.0). Equilibrium dialysis was performed at 5 °C overnight. B, equilibrium binding curves for TRAP binding to (GAGUU)11 RNA. Filter binding analysis of native (f) and renatured (E) TRAP in the presence of 1 mM L-tryptophan, as well as for renatured TRAP in the absence of added tryptophan (●). Renatured TRAP was prepared as described above for A. Each point represents the average of at least two independent experiments. Standard deviations were less than 10% of the mean.

11841

11842

TRAP-RNA Interaction by Subunit Mixing TABLE I RNA binding affinity of wild type and mutant TRAP hetero-11-mer mixtures WT

Mutant

Kd to (GAGUU)11 RNA WT-K71A

%

100 90 80 70 60 40 20 10 5 0

WT-K56A nM

0 10 20 30 40 60 80 90 95 100

2.0 1.5 1.5 1.4 3.5 2.6

1.0 1.1 3.0 3.1 4.3 8.0 15.3 25.5 33 NBa

NB indicates no detectable binding activity up to 5 ␮M mutant TRAP. a

ation method yields fully functional 11-mers when subunits from active proteins are used. We then examined the activities of heteromeric 11-mers composed of wild-type and mutant subunits defective in RNA binding. Our previous studies identified several mutant TRAP proteins that are severely defective in RNA binding but fully functional for tryptophan binding, including K37A, K56A, and R58A (19). Substituting Lys56 with alanine (K56A) is predicted to disrupt 11 hydrogen bonds between (GAGUU)11 and the protein because the Lys56 side chain from each subunit forms a hydrogen bond with O6 of the third G in each GAG repeat in the crystal structure (15). Binding of K56A TRAP to (GAGUU)11 is undetectable using up to 5 ␮M protein, whereas wild-type TRAP binds this RNA with a Kd of 1 nM (Table I). By mixing different ratios of wild-type and K56A TRAP subunits, we generated eight pools of TRAP heteromers (Table I). Each pool contains a different mixture of hetero-11-mers containing between 0 and 11 lysines at position 56, and the abundance of each hetero-11-mer species varies with the input ratio of the two types of monomers based on random assortment. Rather than attempt to separate the different hetero-11-mers, we examined the RNA and tryptophan binding properties of each pool (Table I). All eight pools of hetero-11-mers exhibited similar tryptophan binding properties to wild-type TRAP (S0.5 ⫽ 5–10 ␮M and n ⫽ 1.4 to 1.6; data not shown). In contrast, as the percentage of wild-type subunits in the mixtures decreased from 100 to 5%, the affinity of each pool for (GAGUU)11 RNA decreased from 1 to 33 nM (Table I). Surprisingly, heteromeric mixtures containing only 5 or 10% wild-type TRAP displayed apparent Kd values of 25.5 and 33 nM, respectively (Table I), whereas we observed no detectable binding with up to 5 ␮M K56A homo-11-mer. The hetero-11-mers in these pools are predicted to have an average of ⬃1 wild-type subunit. Hence these results suggest that the presence of very few wild-type subunits in an 11-mer dramatically stabilizes the interaction with RNA as compared with having none. DISCUSSION

Our studies show that GdnHCl reversibly denatures the folded TRAP 11-mer into unfolded monomers. Because the molecular structure of TRAP in the absence of bound tryptophan has not yet been determined, the oligomeric state of the apo-protein has not been conclusively demonstrated. However, the analytical ultracentrifugation studies presented here show that apo-TRAP remains an 11-mer (apparent molecular mass, 93 kDa). This conclusion is further supported by nanospray mass spectrometry studies, which show that in the absence or

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shown to be 11-mers by size exclusion chromatography (data not shown). The K71A 11-mer has much higher mobility on the gel than wild-type TRAP (Fig. 5A, compare lanes 1 and 2). This result is consistent with the larger net negative charge of the K71A protein under these conditions (pH 8.0) because of 11 basic lysines having been replaced with neutral alanines. Comparable results were also observed for other TRAP mutants with similar substitutions (K37A, K56A, and R58A; data not shown). Renatured wild-type (lane 3) and K71A TRAP (lane 8) have mobilities similar to those of their respective native proteins (lanes 1 and 2). When the proteins created by mixing various ratios of wild-type and K71A subunits prior to renaturation are run on the gel, multiple bands are seen, with mobilities intermediate between the wild-type and K71A homo-11mers (lanes 4 –7). Examination of these lanes reveals a total of 12 different bands; two correspond to the wild-type and K71A homo-11-mers, and the 10 other bands are evenly spaced between the two homo-11-mers. Hence we propose that the 12 bands correspond to 11-mers containing different numbers of wild-type and K71A subunits and that the even spacing arises from the difference in mobility caused by a change of one lysine to alanine between adjacent species. When different ratios of wild-type and K71A subunits were mixed, different distributions of band intensities are seen (compare lanes 4 –7). These observations suggest that when denatured wild-type and K71A subunits are mixed and renatured, pools containing various ratios of the 2 homo-11-mers and the 10 possible hetero-11mers of different composition are created. To quantify the relative abundance of each type of 11-mer in the pools, we determined the intensity of each band in this gel by densitometry. If the assembly of each type of subunit into the 11-mers is random, then the composition of each pool of hetero-11-mers should reflect the starting ratio of both subunits. We found a strong correlation between the experimental data and the predicted distribution for random assortment of subunits based on a polynomial distribution (Fig. 5B; p ⬍ 0.001), indicating that wild-type and K71A TRAP subunits assemble randomly into hetero-11-mers. The presence or absence of tryptophan had no apparent effect on subunit mixing (data not shown). We also observed similar distribution patterns for mixing K37A, K56A, or R58A with wild-type TRAP (data not shown). We then tested the concentration of GdnHCl required for subunit mixing by comparing the ratio of homo-11-mers to hetero-11-mers after incubating for 2 h in the denaturant, followed by dialysis and native gel electrophoresis (data not shown). We were unable to see mixing below 0.5 M GdnHCl. There was partial mixing in 0.5–3.0 M GdnHCl, and with ⱖ3.0 M GdnHCl complete random mixing occurred (data not shown). These observations correlate well with our results from size exclusion chromatography and analytical ultracentrifugation (Fig. 3). Hence we conclude that subunit mixing into heteromeric 11-mers occurs only after the TRAP 11-mers are denatured into unfolded monomers and that refolding and random assembly then occur de novo. RNA Binding Activity of Heteromeric TRAP Proteins—In the three-dimensional structure of TRAP, Lys71 is located distant from the tryptophan binding and RNA binding sites (15), and previous studies have shown that the K71A mutant protein is fully functional in vivo (19). We confirmed that the K71A homo11-mer has biochemical properties nearly identical to those of wild-type TRAP in vitro (data not shown). Moreover, we found that all heteromeric mixtures of wild-type and K71A TRAP had affinities and specificities for binding RNA (Table I) and tryptophan (data not shown) similar to those of wild-type TRAP. These studies further confirm that our denaturation/renatur-

TRAP-RNA Interaction by Subunit Mixing

4 5

D. Scott, unpublished results. Y. Chen and P. Gollnick, unpublished observations.

solely between wild-type subunits in the hetero-11-mers and the RNA because the binding of one or two GAG repeats to wild-type TRAP 11-mer is undetectable (29). Moreover, this conclusion is not limited to Lys56 because we obtained very similar results with mixing subunits from other similar RNA binding mutants of TRAP, including K37A and R58A (data not shown). In a recent study, a similar phenomenon was observed when using nucleoside analogs to probe the TRAP-RNA interaction (16). The 2⬘-OH of the ribose on the third G of each (G/U)AG repeat hydrogen bonds to the peptide NH of Phe32 in each TRAP subunit (15). Substituting all 11 of these riboguanosines with deoxyriboguanosine eliminated measurable binding of the resulting chimeric D/RNA to TRAP (16). However, the presence of just such a single riboguanosine in one repeat (with deoxyriboguanosine in the other 10 repeats) dramatically stabilized the complex yielding a Kd of 90 nM. This observation is similar to our findings with hetero-11-mers, in which we noted a dramatic increase in the stability of a complex with pools containing on average 1 wild-type subunit/11-mer (Kd ⫽ 20 –30 nM) as compared with the mutant homo-11-mer (Kd⬎⬎5 ␮M). These observed differences in affinity cannot be attributed solely to the presence of one hydrogen bond in the complex or even to the participation of only one subunit or RNA repeat. We therefore propose that the presence of one fully functional subunit or RNA triplet repeat serves to facilitate the interactions of the remaining mutant subunits or repeats in the complex. This conclusion is supported by RNase protection studies demonstrating that all 11 triplet repeats were protected in D/RNA chimeras that contain only one or two fully functional repeats.6 The position of the deoxyriboguanosine substitutions in the RNA had little effect on the binding affinity to TRAP, suggesting that different arrangements of one or two active subunits in the circular 11-mer is unlikely to significantly affect the affinity for RNA. Elliott et al. (16) proposed a two-step model for RNA binding to TRAP in which binding initiates with a single triplet repeat forming an initiation complex with the protein, followed by cooperative binding of the remaining repeats. Our studies of hetero-11-mers of TRAP further support and extend this model by showing that the initiation complex requires at least one fully active subunit in the protein combined with a fully functional repeat in the RNA. The initiation complex tethers the RNA on the protein, thus restricting its degree of freedom as well as placing the remaining repeats in the proper register to interact with their binding sites on the protein. Hence once the initiation complex has formed, imperfect binding units in either the protein or RNA are tolerated in forming the remainder of the complex. This two-step binding model has implications in the mechanism of TRAP-mediated transcriptional attenuation. During attenuation, TRAP binds the nascent trp leader RNA and affects formation of the transcription terminator, which in turn halts RNA polymerase. This binding must occur within a very short period of time before RNA polymerase passes the terminator. Hence the kinetics of both initiation and propagation of TRAP-RNA interaction relative to the process of transcription elongation are critical for the efficiency of attenuation regulation. Acknowledgments—We thank Sathyamangalam V. Balsubramanian and Gary A. Baker for assistance with instruments. We thank Jim Stamos, Alan Siegel, and Alfred Antson for preparation of figures and Gerald Koudelka, Paul Babitzke, and Charles Yanofsky for critical reading of the manuscript.

6

P. Li and P. Gollnick, unpublished observations.

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presence of tryptophan TRAP exists solely as an 11-mer.4 At ⱖ 3.0 M GdnHCl, CD studies show that the secondary structure of TRAP is unfolded into random coils. In addition, data from analytical ultracentrifugation, fluorescence anisotropy, and size exclusion chromatography demonstrate that the TRAP 11-mer is dissociated into monomers at ⱖ3.0 M GdnHCl. Removing GdnHCl not only refolds the overall secondary structure and oligomerization of TRAP, it also restores the biochemical activities of tryptophan and RNA binding of the protein. Mixing denatured subunits from two different types of TRAP proteins (mutant and wild type) and then removing the denaturant result in random assembly of the subunits into hetero11-mers (Fig. 5A). The observation that assembly of the subunits into 11-mers is random indicates that assembly only occurs with individual subunits. We limited our studies to mutant proteins in which the altered residues are on the surface of the protein and do not interact with neighboring subunits (1), including Lys37, Lys56, Arg58, and Lys71. The observation that subunits from all four of these mutant proteins can randomly assemble with wild-type monomers indicates that none of these residues are critical for the processes of folding and oligomerization. In B. subtilis, wild-type TRAP down-regulates expression of trp genes in the presence of excess tryptophan, whereas mutations that alter residues involved in RNA binding, such as K56A, eliminate regulation (1). Coexpressing increasing levels of K56A and a fixed level of WT TRAP in B. subtilis reduced but did not completely eliminate regulation of the trp genes in response to tryptophan.5 These observations suggest that hetero-11-mers consisting of WT and K56A subunits can also be formed in vivo. We obtained insights into the mechanism by which TRAP binds RNA from our experiments with mixed hetero-11-mers of wild-type and K56A TRAP. The most surprising finding was that very low percentages of wild-type subunits (5–10%) in the mixture dramatically enhanced the observed affinity of the mixed hetero-11-mers for RNA as compared with the K56A mutant protein (Table I). One explanation for these findings could be that the observed RNA binding affinity is due to the presence of a small fraction of wild-type homo-11-mers or hetero-11-mers with large numbers of wild-type subunits within these pools. The measured affinity for any pool is the weighted average of the dissociation constants for each of the TRAP hetero-11-mer species comprising the pool. Moreover, our results show that subunit mixing into these heteromers is based on random assortment, and the composition of each pool is predicted by a binomial distribution (Fig. 5B). Hence, for example, wild-type homo-11-mers (WT11) with a Kd of 1 nM are predicted to be less than 1:1011 or 1:1014 of the populations in the pools made from 90 or 95% K56A (WT11 ⫽ 0.111 or ⫽ 0.0511). Native gel analysis of these pools is consistent with this prediction, because all the visible bands are positioned just above the band for the K56A homo-11-mer (data not shown). If wild-type homo-11-mers were the sole contributor to the observed Kd of the pool with 10% wild-type subunits, then the predicted value would be 100 M (1/1011 of the pool with a Kd of 1 nM). Moreover, we observed an apparent Kd of 33 nM for the 5% WT, 95% K56A mixture (Table I) in which only about 1.5% of the 11-mers have more than two wild-type subunits. Together these findings suggest that TRAP 11-mers with as few as one or two wild-type subunits have relatively high affinity for (GAGUU)11 RNA as compared with the K56A homo-11-mer. The observed affinity is unlikely to be the result of interactions

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