Bacillus subtilis TRAP Binds to its RNA Target by a 5′ to 3′ Directional Mechanism

doi:10.1016/j.jmb.2004.10.071

J. Mol. Biol. (2005) 345, 667–679

Bacillus subtilis TRAP Binds to its RNA Target by a 5 0 to 3 0 Directional Mechanism Maria V. Barbolina, Xiufeng Li and Paul Gollnick* Department of Biological Sciences, State University of New York, Buffalo, NY 14260 USA

TRAP is an 11 subunit RNA-binding protein that regulates expression of the Bacillus subtilis trpEDCFBA operon by transcription attenuation and translation control mechanisms. Tryptophan-activated TRAP acts by binding to a site in the 5 0 -untranslated leader region of trp mRNA consisting of 11 (G/U)AG repeats. We used mung bean nuclease footprinting to analyze the interaction of TRAP with several artificial binding sites composed of 11 GAG repeats in nucleic acids that lack secondary structure. Affinities for individual repeats within a binding site did not vary significantly. In contrast, the association rate constants were highest for repeats at the 5 0 end and lowest for those at the 3 0 end of all binding sites tested. These results indicate that TRAP binds to its RNA targets by first associating with one or more repeat at the 5 0 end of its binding site followed by wrapping the remainder of binding site around the protein in a 5 0 to 3 0 direction. This directional binding is novel among RNA-binding proteins. We suggest that this mechanism of binding is important for TRAP-mediated transcription attenuation control of the trp operon. q 2004 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: RNA-binding protein; footprinting; gene regulation; binding kinetics; tryptophan

Introduction Expression of the tryptophan biosynthetic genes in Bacillus subtilis is regulated in response to changes in intracellular levels of L-tryptophan by an RNA-binding protein called TRAP (trp RNAbinding attenuation protein).1,2 TRAP regulates transcription of the trpEDCFBA operon through an attenuation mechanism that controls the formation of two mutually exclusive RNA secondary structures, an intrinsic terminator and an antiterminator, in the 203 nt untranslated leader region 5 0 to trpE (Figure 1).3 In the presence of excess tryptophan, TRAP is activated to bind to the nascent trp leader transcript. This binding induces formation of the terminator structure thus halting transcription in the leader region and preventing expression of the trp genes.4 In limiting tryptophan, TRAP does not Present address: Maria V. Barbolina, Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA. Abbreviations used: MBN, mung bean nuclease; ka, association rate constants. E-mail address of the corresponding author: [email protected]

bind RNA, the antiterminator structure forms, and the trp operon is transcribed. In addition, TRAP regulates translation of several trp genes by either altering RNA secondary structure or through direct competition with ribosomes for binding to the mRNA.5–10 TRAP is composed of 11 identical subunits arranged in a ring structure and is activated to bind RNA by binding up to 11 molecules of L -tryptophan in pockets between adjacent subunits.11,12 NMR studies suggest that tryptophan binding activates TRAP, at least in part, by reducing the flexibility (dynamics) of the protein.13 TRAP binds to RNAs that contain multiple NAG repeats separated from each other by several non-conserved “spacer” nucleotides.14,15 The binding site in the trp leader region contains 11 (G/U)AG repeats; the first five of which are in a single-stranded region of the RNA (Figure 1). Structures of TRAP complexed with RNAs containing 11 GAG,16,17 or 11 UAG18 repeats show that the single-stranded RNA wraps around the outside of the protein with the phosphodiester backbone of the bound RNA on the outside of the ring and the bases pointing inward. The majority of the contacts between the protein and RNA are to the bases of the (G/U)AG

0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.

668

The Kinetics of TRAP Binding to RNA

Figure 1. Model of transcription attenuation of the B. subtilis trp operon. Large boxed letters designate the complementary strands of the terminator and antiterminator RNA structures. TRAP is shown as a ribbon diagram with each of the 11 subunits in various colors and the bound RNA shown forming a matching circle upon binding to TRAP. The GAG and UAG repeats involved in TRAP binding are shown in ovals and are also outlined in the sequence of the antiterminator structure. Numbers indicate the residue positions relative to the start of transcription. Nucleotides 108–111 overlap between the antiterminator and terminator structures, and are shown as outlined letters.

repeats.16–19 The only direct contact with the RNA backbone is a hydrogen bond with the 2 0 -OH group of the ribose on the third G residue of each repeat. The remaining residues in the TRAP binding site can be replaced with deoxyribonucleotides, and the DNA/RNA chimera (taGcc)11†, binds to TRAP with equal affinity and specificity as the corresponding RNA (UAGCC)11.20 Moreover, chimeras that contain ribose in only the third position (riboG3) of as few as four of the 11 repeats bind TRAP with only five- to tenfold lower affinity than the corresponding RNA.21 These and other studies led us to propose a two-step model for RNA binding to TRAP.21,22 In the first step an initiation complex forms between one and two trinucleotide repeats in the RNA and binding sites on TRAP. The second binding step involves wrapping the remaining RNA repeats around the protein. Previous studies of RNA binding to TRAP have focused on equilibrium binding thus yielding little † To simplify designation of chimeric DNA/RNA sequences, deoxyribonucleotides are represented in lower case (g, a, t and c), and ribonucleotides are represented in upper case (G, A, U and C).

information about the kinetic parameters of this interaction. Moreover, while several studies have examined TRAP binding to RNAs that contain fewer than 11 triplet repeats,14,20,21 binding to individual repeats within an RNA containing multiple repeats has not been investigated. Here, we used rapid quench mung bean nuclease footprinting to analyze the equilibrium and kinetic properties of TRAP binding to several nucleic acids that contain 11 GAG repeats. The advantage of this footprinting method is that we can examine binding to individual repeats within the binding sites. We examined binding to one RNA and to several DNA/RNA chimeras composed mainly of deoxyribonucleotides except for various numbers of ribonucleotides in the third residue (riboG3) of the repeats. In all cases there was no predicted secondary structure in the binding sites. We found that equilibrium binding affinities (Kd) of the individual repeats for TRAP did not vary significantly within the binding sites. In contrast, for all four binding sites, TRAP bound most rapidly to the triplet repeats at the 5 0 end of the binding site and slowest to those at the 3 0 end. These results suggest that when TRAP binds RNA, it forms an

The Kinetics of TRAP Binding to RNA

initial complex with one or more triplet repeat(s) at the 5 0 end of its binding site followed by wrapping the remainder of the binding site in the 5 0 to 3 0 direction.

Results Mung bean nuclease footprinting: equilibrium binding We developed a nuclease protection (footprinting) assay to examine the mechanism by which the TRAP 11mer binds to individual (G/U)AG trinucleotide repeats within an RNA target consisting of 11 repeats. We chose mung bean nuclease (MBN) because this endonuclease cleaves only singlestranded RNA or DNA,23 which allowed us to examine TRAP binding to both RNA and DNA/ RNA chimeras. The RNA and DNA/RNA chimeras used in this study contain an 11 GAG repeat TRAP binding site flanked by 60–110 residues on both the 5 0 and 3 0 side of the binding site (Figure 2). We designed these nucleic acids to contain little or no predicted secondary structure for three reasons. First, secondary structure inhibits cleavage by MBN. 23 Second, secondary structure inhibits TRAP binding to its binding site.14,15,24 Third, it is unknown whether secondary structure in the flanking regions could influence the mechanism of TRAP binding. The 11 GAG repeat TRAP binding site in all four nucleic acids we studied are predicted to be unstructured.25 The only predicted secondary structure in any of the regions that flank the TRAP binding site is a small stem–loop (predicted TmZ30 8C) 20 nt downstream of the TRAP binding site in the three DNA/RNA chimeras; there is no predicted secondary structure in the flanking regions of (GAGUA)11 RNA. We first examined equilibrium binding of TRAP to an RNA containing 11 GAGAU repeats (Figure 2A). MBN cleaves single-stranded DNA or RNA after all four nucleotides but with preferences

669 for AOTOCOG.26 These preferences are apparent in the cleavage pattern of (GAGUA)11 RNA such that digestion conditions yielding uniform cleavage of the GAGAU repeats in the TRAP binding site resulted in slight over-digestion of an A-rich region immediately after the binding site (labeled*) and slight under-digestion of a G-rich segment in the 5 0 flanking region (between nucleotides 70 and 80) (Figure 3A). Adding tryptophan-activated TRAP resulted in protection of the residues within the binding site, whereas those in the flanking regions were not protected (Figure 3A). Although there appears to be slight protection of short G-rich segment in the 5 0 flanking region as well as the Arich segment in the 3 0 flanking region (Figure 3A), these observations were not reproducible in all gels. The maximal fractional protection for the entire binding site was z0.34 and varied little between individual repeats (Figure 3B); we observed similar fractional protection of 0.3–0.4 for all binding sites tested in this study. The observed maximal protection of 0.34 under conditions (50 nM TRAP) where we have shown that all the RNA is complexed with TRAP,20,21 likely reflects that binding to TRAP only reduces the efficiency by which MBN cleaves the bound RNA as compared to the unbound regions of RNA rather than fully protects the RNA from MBN. This conclusion is consistent with RNA binding to the outside of TRAP with the phosphodiester backbone on the outside of the complex.16–19 Moreover, although the crystal structures show that the spacer nucleotides between the (G/U)AG repeats do not directly interact with TRAP,16–19 the AU residues between the GAG repeats of (GAGUA)11 were protected from MBN digestion equally as well as the GAGs (Figure 3A). Similar protection of the spacer residues has also been observed in other nuclease footprinting studies.9,27 Together these observations suggest that TRAP protects bound RNA by steric hindrance of MBN. By examining the fractional protection of the 11 repeat binding site as a function of TRAP concentration we derived a binding curve (Figure 3C) and

Figure 2. Schematic representation of the nucleic acids used in the study. The TRAP binding sites are shown in boxes, and the 5 0 and 3 0 flanking sequences are shown schematically. A, (GAGUA)11 RNA. B, DNA/RNA chimeras. Sequence of the TRAP binding site is shown for each of the chimeras in the order 5 0 FourRiboG, 3 0 FourRiboG, ElevenRiboG from the top to the bottom. Ribonucleotides are represented in upper case and deoxyribonucleotides are in lower case letters. The entire sequences of each nucleic acid are in the Supplementary Material, Table 2.

670

The Kinetics of TRAP Binding to RNA

Figure 3. A, Representative mung bean nuclease footprint of TRAP binding to 32P-labelled (GAGUA)11 RNA (5 pM). TRAP concentration was increased from 0 nM to 8 nM (note only a subset of the total lanes on the gel (0–50 nM) are shown for simplicity. The position of the TRAP binding site is indicated by a bracket on the left side of the gel with 11 small brackets indicating the positions of individual GAG repeats, numbered from the 5 0 end of the binding site. Sequences preceding the TRAP binding site are designated as 5 0 flank and those following the TRAP binding site are designated 3 0 flank with an asterisk (*) indicating an A-rich region. The locations of several size standards are indicated on the right side of the gel. B, Fractional protection (P) as defined by equation (1) (Experimental Procedures) of the individual GAGAU repeats within the TRAP binding site in the absence (black bars) and in the presence (gray bars) of 50 nM TRAP. The region designated 5 0 flank corresponds to residues 50–80, and 3 0 flank corresponds to residues 136–170. The bars represent the average of seven independent experiments with standard error of 7% of the mean. C, Equilibrium binding curve for TRAP binding to (GAGUA)11 RNA. Data are the average of seven independent experiments, with standard error of 7% of the mean.

a dissociation constant (Kd) of 1.0 nM for TRAP binding to (GAGUA)11. This value is in excellent agreement with the Kd of 1.1 nM for TRAP binding to this RNA determined by filter binding.20,21 In all cases we analyzed the footprinting data based on a stoichiometry of 1 TRAP 11mer binding to each RNA molecule. This stoichiometry is consistent with the crystal structures of several TRAP–RNA complexes.16–19 In addition we used mobility-shift gels to demonstrate that under conditions similar to

those we used in these footprinting studies the stoichiometry is 1:1 (See Supplementary Material, Figure 1). TRAP also binds to DNA/RNA chimeras composed mainly of deoxyribonucleotides, with ribonucleotides in only the third residue (riboG3) of the (G/U)AG repeats.20,21 MBN footprinting showed that TRAP protected the 11 gaGtt repeats of the binding site of ElevenRiboG, whereas the flanking sequences were not protected (not shown but see

The Kinetics of TRAP Binding to RNA

671

Figure 4. Kd values for individual repeats for TRAP binding as determined by protection from mung bean nuclease. A, 5 0 FourRiboG DNA/RNA chimera; B, The 3 0 FourRiboG DNA/RNA chimera; C, ElevenRiboG DNA/RNA chimera; and D, (GAGUA)11 RNA. Repeats are numbered 1–11 from the 5 0 end of the binding site. RiboG3-containing repeats are represented as filled circles (C), repeats consisting of only deoxyribonucleotides are represented as open circles (B). Data are the average of at least four experiments with standard errors shown. Kd values for individual repeats within all four binding sites are in given Supplementary Material, Table 3.

Figure 5A). The DNA residues of the gaGtt repeats were protected equally as well as the riboG3 residues. Analysis of protection of the entire binding site yielded a Kd of 1.4 nM, which agrees well with the value of 0.5 nM determined.21 These results confirm that TRAP binds to a DNA/RNA chimera with ribose in only the third residue of each repeat by a similar mechanism and with affinity comparable to that of RNA of analogous sequence. Equilibrium footprinting studies of DNA/RNA chimeras containing riboG3s in only the first four repeats (5 0 FourRiboG; Figure 2B) or in the last four repeats (3 0 FourRiboG; Figure 2B) yielded similar protection patterns as those seen with ElevenRiboG. In both cases, all 11 repeats in the binding site were protected from cleavage approximately equally well even though only four repeats contained riboG3 residues and the remaining seven were entirely DNA. Analysis of the protection of the binding sites yielded Kd values of 3.3 nM and 4.9 nM for 5 0 FourRiboG and 3 0 FourRiboG, respectively, which agree well with the Kd of 5.1 nM obtained previously for a DNA/RNA chimera with four riboG3s.20 We found only small differences in Kd values for the individual repeats within any of the binding sites (Figure 4A–D). Not the absence or the presence of riboG3s in the repeats, the location of the repeats within the binding site, or the nature of the flanking sequences consistently affected the equilibrium binding affinities of TRAP for individual repeats.

Kinetics of TRAP binding: examining the entire binding site To examine the kinetic properties of TRAP binding to RNA or DNA/RNA chimeras, we performed protection assays under pre-steady state conditions. Figure 5A shows a time-resolved MBN footprinting gel for TRAP binding to the DNA/RNA chimera 5 0 FourRiboG. The cleavage pattern of this DNA/RNA chimera appears slightly different from that seen for (GAGAU)11 RNA (Figure 3A). These differences reflect the preferences of MBN for dA residues over dT, dC or dG, as well as for riboG residues (labeled C) as compared to dG residues (labeled K); all of which have been reported.28 Residues within the binding site were protected from digestion by TRAP, whereas the flanking sequences were not. In some cases, repeats at the 5 0 end of the binding site were slightly more protected than those at the 3 0 end. By fitting the fractional protection of the entire binding site as a function of time (Figure 5B) to equation (3) (Experimental Procedures), we obtained observed rate constants (kobs), from which the association rate constants (ka) were derived using equation (4). For each nucleic acid, we determined kobs at several different concentrations of TRAP (5–300 nM) and found the values to be directly proportional to the protein concentration (Figure 5E). The ka values presented in Table 1 are averages derived from experiments

672

The Kinetics of TRAP Binding to RNA

Figure 5. A, Mung bean nuclease footprint of 70 nM TRAP binding to 5 0 FourRiboG DNA/RNA chimera (50 pM). Incubation time of TRAP with RNA from 0 to 45 seconds is indicated. The position of the TRAP binding site is shown by a bracket on the left side of the gel, with 11 small brackets showing positions of the individual trinucleotide repeats. RiboG residues (C) and deoxyG residues (K) within the TRAP binding site are indicated. 5 0 flank and 3 0 flank indicate sequences preceding and following the TRAP binding site. B, Kinetic binding curve for TRAP binding to the entire binding site of 5 0 FourRiboG DNA/RNA derived from the gel presented. C, Kinetic binding curves for TRAP binding to the first (C) and the last (11th) (B) repeats of the binding site on derived from the gel presented. Fractional protection of the 11th repeat was twice lower than that for the first repeat and was normalized to simplify visualization. D, Association constants (ka) for TRAP binding to individual repeats in 5 0 FourRiboG. Repeats are numbered from the 5 0 end of the binding site. RiboG3-containing repeats are represented as filled circles (C) and repeats consisting of only deoxyribonucleotides are represented as open circles (B). Values for association rate constants for individual repeats within the binding site are given in Supplementary Material, Table 4. E, Plot of the dependence of the observed rate constants (kobs) for TRAP binding to its binding site in 5 0 FourRiboG as a function of the concentration of TRAP in the experiment.

673

The Kinetics of TRAP Binding to RNA

Table 1. Association rate constants of TRAP binding to the entire binding site of investigated DNA/RNA chimeras and RNAs DNA/RNA chimera or RNA

Nucleotides in 5 0 flanking region

ka!106 MK1 sK1

15 80 110 110 110 150

10.0G1.3 7.0G2.3 4.5G1.3 3.7G1.3 3.5G2.3 3.0G0.14

ElevenRiboG 5 0 short (GAGAU)11 RNA ElevenRiboG 5 0 FourRiboG 3 0 FourRiboG (GAGAU)11 EX RNA Data are average of at least seven experiments.

using two to four different protein concentrations, each done in at least duplicate. These values are derived from a single-step binding model (see Experimental Procedures). We also evaluated whether the data fit better to a two-step binding model: YZYmax1(1K(KK1X))CYmax2(1K(KK2X)). We found that the data did not fit better to this model and hence have used the simpler one-step model to evaluate all the kinetic data presented here. Kinetics of TRAP binding to individual repeats within binding sites Based on our previous studies we have proposed a two-step model for RNA binding to TRAP in which an initiation complex first forms between one or more triplet repeats in the RNA and the protein, and subsequently the remaining triplet repeats wrap around the protein ring.12,21 Our previous data suggest that formation of the initiation complex requires interaction between at least one fully functional triplet repeat in the RNA and WT binding site on the protein.12,20–22 In contrast, after the initiation complex has formed, the second binding step can proceed even if the RNA or TRAP subunits lack one of the functional groups that interact in the complex.12,20–22 We expected that if an RNA or DNA/RNA chimera contained 11 identical repeats, TRAP could initiate binding randomly at any of the repeats. If true, this randomization would make analysis of kinetic footprinting for the individual repeats within a binding site impossible, because we would only observe an overall averaged protection for all 11 repeats. To attempt to overcome this potential problem, we considered our observations that TRAP binds to DNA/RNA chimeras with as few as four riboG3-containing repeats with only slightly lower affinity than the corresponding RNA, whereas there is no detectable binding to the corresponding DNA sequence entirely lacking riboG3s.21 Based on these findings we hypothesized that the ribose in the third residue of the (G/U)AG repeats might be required to form the initiation complex with TRAP. If so, we would be able to control where TRAP initiates binding to an 11 repeat DNA/RNA chimera by where we place the riboG3s, and so be able to follow the mechanism of binding using time-resolved footprinting. We first examined TRAP binding to the

DNA/RNA chimera 5 0 FourRiboG in which only the first four of the 11 repeats of the TRAP binding site contain riboG3s (Figure 5A). The association rate constants (kas) were greatest for the riboG3containing repeats 1–4 at the 5 0 end of the binding site and were lower toward the 3 0 end of the binding site with repeat 11 binding the smallest (Figure 5C and D). There was approximately a 2.5-fold difference in kas between the first repeat (kaZ2.7! 106 MK1 sK1) and the last repeat in the binding site (kaZ1.1!106 MK1 sK1). These observations suggest that TRAP initiates binding at the 5 0 end of the 11-repeat binding site followed by binding to the 3 0 repeats. To test whether the riboG3 residues are important for initiation of TRAP binding, we examined binding to 3 0 FourRiboG, in which the four riboG3containing repeats are located at the 3 0 end of the binding site (Figure 2B). If the location of the ribosecontaining repeats determines where TRAP initiates binding, then we would predict that in this case the ka values would be greatest for repeats 8–11. Surprisingly, the association rates for TRAP binding to this chimera were not fastest for the riboG3-containing repeats. Instead, we again found that repeats 1–3 at the 5 0 end of the binding site showed the fastest binding to TRAP, then decreased toward the 3 0 end of the binding site and were slowest for the last three repeats (Figure 6A). These results indicate that, as was observed for 5 0 FourRiboG3, TRAP binds first to the 5 0 -most repeats in 3 0 FourRiboG and then wraps the remainder of the binding site around the protein in the 5 0 to 3 0 direction. The results presented above suggest that the location of the riboG3-containing repeats does not influence the kinetic mechanism of TRAP binding. To test this possibility, we examined TRAP binding to ElevenRiboG, which contains 11 identical gaGtt repeats all with riboG in the third residue. Again, TRAP bound to repeats 1–3 at the 5 0 end of the binding site with the greatest association rate constants and with slower rates for the repeats toward the 3 0 end (Figure 6B). Together these results indicate that TRAP initiates binding to the 5 0 -most repeats in all three DNA/RNA chimeras, regardless of the number and location of the riboG3-containing repeats. We then investigated TRAP binding to (GAGUA)11 RNA, which contains 11 identical

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The Kinetics of TRAP Binding to RNA

GAGAU repeats. As was observed for all three chimeras, the fastest rates for TRAP binding were for the repeats at the 5 0 end of the binding site, followed by a decrease in the association rates for repeats toward the 3 0 end (Figure 6(C)).

Discussion Directional binding of TRAP to its binding site

Figure 6. Association rate constants (ka) for TRAP binding to individual repeats in (A) 3 0 FourRiboG DNA/RNA chimera, (B) ElevenRiboG DNA/RNA chimera, and (C) (GAGUA)11 RNA. Repeats are numbered from the 5 0 end of the binding site. RiboG3containing repeats are represented as filled circles (C), and repeats consisting of only deoxyribonucleotides are represented as open circles (B). Values for association rate constants for individual repeats within all three binding sites are in Supplementary Material, Table 4.

TRAP is unique among characterized RNAbinding proteins-in that it is composed of 11 identical subunits arranged symmetrically in a ring structure and that it binds to sites in RNA composed of multiple small repeated elements.16–19 We developed a mung bean nuclease protection assay to examine the individual triplet repeats within a target site during the binding process. There were no significant variations in equilibrium binding affinities between the individual repeats within any of the binding sites we tested. Examining the kinetics of TRAP binding to three DNA/ RNA chimeras and to (GAGUA)11 RNA showed that for all four binding sites the association rate constants were greatest for one or more of the repeats located at the 5 0 end of the binding site and lowest for the 3 0 most repeat(s) (Figures 5D and 6A–C). These variations could not be explained by chance based on Spearman’s correlation for ranked data (P!0.01) and hence suggest that in all four cases, TRAP initiates binding at the 5 0 end of the binding site and then proceeds toward the 3 0 end. This directional (5 0 to 3 0 ) binding was surprising, especially for targets with 11 identical repeats, and to our knowledge is unprecedented among RNAbinding proteins. We therefore considered several factors that potentially could have artifactually influenced our observation of directional binding. First, there was no correlation between the affinity of the individual triplet repeats for TRAP and the order in which TRAP bound to the repeats within a binding site (compare Figures 4A–D with 5D and 6A–C). Second, there was no relationship between the number or location of riboG3-containing repeats and the order of binding. The results with 3 0 FourRiboG were particularly surprising, since we had previously shown that at least one riboG3containing repeat is required for detectable equilibrium binding to TRAP.21 Hence, the role of the riboG3 residues in TRAP binding to DNA/RNA chimeras is not related to forming the initiation complex, suggesting that the riboG3-requirement for stable binding is related to dissociation (off rate). We did attempt pre-steady state footprinting of TRAP on an all DNA oligonucleotide containing 11 GAGTT repeats (no riboG3s) but we were unable to observe any protection of the repeats with up to 500 nM TRAP (higher levels lead to non-specific binding). Third, we considered whether the sequences that flank the binding site in our RNA and DNA/RNA

The Kinetics of TRAP Binding to RNA

chimeras could influence TRAP to initiate binding at the 5 0 end of each site. The flanking regions are the same in all of our DNA/RNA chimeras and consist entirely of DNA sequences, mainly equivalent to several segments of the B. subtilis trp leader region (Figure 2A). The flanking sequences in (GAGUA)11 RNA are different from the chimeras both in terms of composition, which is entirely RNA, and sequence, which is mostly poly(A) (Figure 2B). Given these differences it seems unlikely that effects of the flanking sequences explain the directional binding that we observed for all four targets we tested. Finally, secondary structure in either binding site or flanking sequences might influence the observed directional TRAP binding. However, (GAGUA)11 RNA is predicted to be entirely unstructured and the only predicted secondary structure in the three DNA/RNA chimeras is a small stem–loop downstream of the binding site. Therefore, the observed 5 0 to 3 0 binding of TRAP is not readily explained by the presence of secondary structure. These findings raise the question: how does TRAP first recognize the 5 0 most triplet repeat(s) among 11 identical repeats in its binding site? One possible mechanism would be for TRAP to first associate with the 5 0 end of the RNA and then diffuse along the RNA until it encounters one or more G/UAG repeat(s). If true, then the rate of binding to the first repeat of a binding site should depend on the distance between the 5 0 end of the RNA and the (G/U)AG repeat. Consistent with this proposal, we found that for the nucleic acids that we have examined, the association rate constants for TRAP binding to the 5 0 most repeats are directly proportional to the length of the 5 0 flanking segment (Table 1). We considered two models for TRAP recognition of its binding site. First, we applied the diffusiondriven mechanism of protein translocation on nucleic acids. This mechanism is based on the assumption that interactions occur by random collisions. To examine whether our data are described by this mechanism we used the Debye– Smoluchowski equation to calculate the maximum value of ka, the diffusion-limited second-order association rate constant: ka ¼ 4pkfelec bðDtrap þ Dma ÞNo =1000 where k is a steric interaction factor corresponding to the fraction of the total collisions that are successful, felec is an electrostatic factor, b is the interaction radius for the reaction, Dtrap and Drna are the three-dimensional diffusion constants for TRAP and RNA, and No is Avogadro’s number. Using previously determined values for these par˚ ,16 we obtain an absolute ameters29 and Dtrap of 80 A maximum value of kaz109 MK1 sK1. Hence, if the diffusion model is true for the TRAP–RNA complex, the value of the association rate constant can range from zero to 109 MK1 sK1. The association rate values we determined by MBN footprinting are

675 10(G3)!106 MK1 sK1, which fall within the allowable range set by the diffusion-driven model. The second mechanism that potentially could apply to our system is the “faster-than-diffusioncontrolled” interaction previously observed for Escherichia coli lac repressor–operator complex.29 This model is designed to explain DNA–protein interactions that occur with higher than the diffusion-driven rates. This mechanism is not necessary to describe the interaction of TRAP with RNA, since the ka values we measured are lower then the maximum for the diffusion model. Our findings also suggest that after TRAP initiates binding to one or more repeats at the 5 0 end of the binding site, it then wraps the reminder of the binding site around the protein in the 5 0 to 3 0 direction. The affinity of TRAP for RNA increases as the number of repeats increase,21 suggesting a simple driving force for TRAP binding to each successive repeat. There are several examples of proteins that function by proceeding directionally along nucleic acids. Among these are RNA and DNA polymerases that synthesize nucleic acids from their 5 0 ends and move along the DNA template from 3 0 to 5 0 . Transcription factor Rho binds to rut sites and then uses ATP to translocate 5 0 to 3 0 along the RNA until it reaches the transcription complex.30–33 Eukaryotic ribosomes also bind to mRNAs at their 5 0 end and then scan in the 5 0 to 3 0 direction until the encounter an AUG start codon. All of these proteins appear to function through different mechanisms than TRAP. Implications of the kinetics of TRAP binding for in vivo function. TRAP regulates transcription of the trp operon by binding to and altering the secondary structure of the leader region of nascent trp mRNAs so as to promote formation of an intrinsic transcription terminator, which halts transcription prior to the structural genes. Implicit in this mechanism is the need for TRAP to bind and alter the RNA structure before RNA polymerase proceeds beyond the terminator sequence. The kinetic properties of TRAP binding are therefore important for its role in transcription attenuation. The 5 0 to 3 0 binding mechanism that we describe here for TRAP binding to several nucleic acids with 11 GAG repeats seems particularly appropriate for TRAP’s function in attenuation. Preliminary studies of an RNA containing the 11 (G/U)AG repeat TRAP binding site from the B. subtilis trp leader region (nucleotides 36–92), which lacks predicted secondary structure,25 indicate that TRAP also initiates binding to the 5 0 -most repeats followed by moving toward the 3 0 end of this site (X.L., M.B. & P.G., unpublished results). We have also shown that TRAP binds to RNAs with as few as four GAGs or UAGs with Kd values below 100 nM,21 and we have estimated the in vivo concentration of TRAP in B. subtilis to be approximately 200 nM.34 Together these observations suggest that TRAP could bind stably to

676 the nascent trp leader RNA when as few as four G/ UAG repeats are available. This situation would occur when RNA polymerase reaches approximately nucleotide 70 in the trp leader region. This estimate is based on 14–15 nt of RNA at the 3 0 end of the transcript being contained in either the DNA/ RNA hybrid or in the exit channel of RNA polymerase and hence, are unavailable for TRAP binding to the nascent leader RNA.35 Therefore, to induce termination, TRAP must bind and alter the trp leader RNA structure in the time it takes RNA polymerase to proceed from residue 70 to 140 (end of the terminator segment; Figure 1). The rate of elongation for bacterial RNA polymerase in vivo has been estimated to be 40–50 nt/s.36–38 Together these approximations suggest that TRAP would have approximately 1.4–1.7 seconds to bind in order to cause termination. Based on the association rate constant we determined for binding to (GAGUA)11 RNA, at 200 nM TRAP (in vivo) the rate of binding would be w1.4 sK1 (7!106 MK1 sK1!200!10K9 M). Hence, these kinetic properties suggest TRAP should be capable of binding and performing its task within the necessary time-frame. In addition Babitzke and co-workers have shown that B. subtilis RNA polymerase pauses at U107 in vitro.39 If this pausing occurs in vivo it would provide additional time for TRAP to bind. An additional conserved feature of the trp leader mRNA is a stem–loop structure at the 5 0 end of the transcript, just upstream of the first triplet repeats in the TRAP binding site.1 We are currently investigating whether the presence of this structure influences the TRAP binding mechanism. One curious aspect of this binding mechanism is that TRAP binds to several RNA targets that are contained in the middle of polycistronic mRNAs, including trpG and ycbK.1 It is difficult to envision that TRAP binds to these targets via this 5 0 to 3 0 scanning mechanism. In all of these cases TRAP regulates translation of the adjacent gene and hence the timing of binding may be less critical. Hence TRAP may bind to these target by a different mechanism. Our data suggest the following sequence of events leading to B. subtilis trp operon termination. When the nascent trp transcript, containing sufficient (G/U)AG repeats for TRAP binding (approximately four) appears from the transcriptional bubble, tryptophan-activated TRAP initiates binding. As additional repeats appear when the enzyme moves on the template DNA, TRAP continues binding the nascent mRNA in the 5 0 to 3 0 direction and prevents formation of the antiterminator (Figure 1), thus leading to termination of transcription within the leader region.

Experimental Procedures Materials All DNA/RNA chimeric oligonucleotides (Dharmacon) and DNA oligonucleotides (Integrated

The Kinetics of TRAP Binding to RNA

DNA Technologies) used in this study are listed in Supplementary Material, Table 1. TRAP was purified as described.40,41 Construction of plasmid pTZGAGAUpolyA All plasmids were propagated in E. coli JM107. Plasmid pTZGAGAUpolyA for in vitro synthesis of the 196 nt (GAGAU)11polyA RNA (called (GAGUA)11 for simplicity) has 11 repeats of the sequence 5 0 -GAGAU-3 0 flanked by 55 A residues and several G residues (Figure 2A). Eleven C residues were placed immediately downstream of the T7 promoter to allow radioactive labeling of the RNA at the 5 0 end by in vitro transcription with T7 RNA polymerase using [a-32P]CTP. The insert was created by a three-way ligation of oligonucleotides #1, #2 and #3 (Supplementary Material, Table 1) using a previously published procedure42 using oligos #4 and #5 as splints in the ligation reaction to ensure that oligos #1, #2 and #3 were ligated in the proper orientation. The resulting single-stranded DNA was converted to double-stranded by the polymerase chain reaction (PCR) using oligonucleotides EcoPrimer and K40 universal as primers, then digested with EcoRI and HindIII, and ligated into similarly cut pTZ18U (USB). Synthesis and labeling DNA/RNA chimeras and RNA The DNA/RNA chimera 5 0 FourRiboG consists of 271 deoxyribonucleotides and four ribonucleotides was synthesized by a three-way ligation42 of oligonucleotides 5 0 FootEnd, FourRiboG and 3 0 100End (Supplementary Material, Table 1) using Clamp4 and Clamp3 as joining oligonucleotides. 5 0 FourRiboG consists of 75 deoxyA residues followed by the DNA sequence corresponding to the B. subtilis trp leader region from C1 to C35 followed by the artificial TRAP binding site (gaGtt)4(gagtt)7 (Figure 2B). Only the first four of the 11 repeats contain a ribose sugar on the residue in the third position (riboG3), whereas the remainder of the TRAP binding site is entirely DNA. The TRAP binding site is followed by the DNA equivalent of trp leader sequence from nucleotides C92 to C184 followed by a binding site for the K40 universal sequencing primer. DNA/RNA chimeras 3 0 FourRiboG and ElevenRiboG were synthesized by the same procedure as above except that oligonucleotides FourRiboG2 and 11RiboG were used instead of FourRiboG in the three-way ligation reactions, respectively. 3 0 FourRiboG and ElevenRiboG have the same sequence as 5 0 FourRiboG except that the ribose containing residues in the TRAP binding sites are arranged differently (Figure 2B). 3 0 FourRiboG contains riboG3s in only the last four repeats, whereas ElevenRiboG contains riboG3 residues in all 11 repeats of the binding site. DNA/RNA chimeras were radioactively labeled at their 5 0 end using [g-32P]ATP (Perkin Elmer) and T4 polynucleotide kinase (USB).43 (GAGUA)11 RNA was synthesized and radioactively labeled by in vitro transcription with T7 RNA polymerase and [a-32P]CTP as described44 using HindIII linearized pTZGAGAUpolyA as template. Mung bean nuclease protection assays Steady-state MBN footprinting experiments were performed using a modification of a previously published procedure for DNase I footprinting.45,46 TRAP (0–70 nM) was incubated with %5.0 pM (5 0 32P)-labeled DNA/RNA

677

The Kinetics of TRAP Binding to RNA

chimera or RNA at 37 8C until equilibrium binding was reached (Rten minutes). Binding reactions (100 ml) contained 16 mM Hepes, (pH 8.0), 250 mM potassium glutamate, and 1 mM L-tryptophan. Nuclease digestion was initiated by adding 0.1 unit of MBN in 100 ml as well as zinc chloride to 1 mM final concentration, and the reaction mixtures were incubated at room temperature (20 8C) for three minutes. Nuclease digestion was stopped with 750 ml of freshly made 0.3 M ammonium acetate (pH 7.0), 2 mM EDTA and 10 mg/ml of yeast tRNA in ethanol. The samples were incubated at K20 8C for at least one hour. Mock-treated control samples were incubated in the absence of MBN. Pre-steady state experiments were also performed using a modification of a previously published procedure for DNase I footprinting.47 TRAP (5.0 to 50 nM) was incubated with %10 pM (5 0 -32P)-labelled DNA/RNA or RNA for 1–300 seconds at room temperature (20 8C) in 16 mM Hepes (pH 8.0), 250 mM potassium glutamate, and 1 mM L-tryptophan. MBN digestion and quenching were performed as described above. For all footprinting experiments the precipitated samples were collected by centrifugation in a microfuge for 15 minutes, washed with 70% ethanol, dried, and resuspended in loading buffer composed of 50% formamide, 10 mM EDTA, 0.025% bromophenol blue, 0.025% xylene cyanol FF. Samples were loaded onto 8% (w/v) polyacrylamide/8 M urea gels, and run at 1300 V for five to six hours, fixed in 20% ethanol, dried and exposed to Molecular Dynamics storage phosphor screens. Data analysis

(1)

Where I is the relative intensity, n refers to any lane containing TRAP, ‘r’ refers to a reference lane without protein, ‘site’ denotes TRAP binding site(s), ‘std’ denotes a standard region of the gel used to correct for variations in loading each lane, which was residues 135–170 for the RNAs and for 165–250 for the chimeras. To obtain binding constants (Kd) the data were fit to equation (2) using GraphPad Prism (GraphPad Software): P Z Pmax ½TRAP
=ðKd C ½TRAP
Þ

P ¼ Pmax ð1 K eKkobs!t Þ

(3)

where Pmax is the maximal fractional protection, t is time. Values for association rate constants (ka) were calculated from the equation: kobs Z ka ½TRAP


(4)

In each case we determined kobs and its standard error at different concentrations of TRAP, derived values of ka and averaged them.

Acknowledgements The authors thank Charley Yanofsky, Paul Babitzke and Jerry Koudelka for critical reading of the manuscript. We thank Amanda Manfredo for excellent technical assistance. We thank Derek Taylor for assistance with statistical analysis. This work was supported by grants GM62750 from the National Institutes of Health and MCB 9982652 from the National Science Foundation.

Supplementary Data

Digital images of the gels were obtained using a Phosphorimager (Molecular Dynamics). Quantification of band intensities and analysis of protection were performed using Image Quant software using procedures developed by Brenowitz and co-workers.45,46 Band intensities for either the entire 11-repeat binding site or for individual repeats within the site were determined by quantifying the absorbance values within a contour defining the corresponding bands. The entire 11-repeat binding site was defined as a rectangle including all 55 bands from the residues of the binding site. Individual repeats within the entire site were defined by rectangles including the five bands from the GAGNN residues of repeat. The location of bands corresponding to the residues of the binding site was determined based on size markers. Since the pre-steady state experiments were performed at [TRAP][[RNA] or [DNA/RNA], the rate of dissociation of TRAP from the RNA (or DNA/RNA) was negligible as compared to the association rate. Hence, we used pseudo-first-order approximation for analysis of the kinetic binding curves. Values for “fractional protection” (P) of the binding sites were obtained by densitometric analysis of the digital images according to a described procedure45,46 using the equation: P Z 1 K ½ðIn;site =In;std Þ=ðIr;site =In;site Þ


where Pmax is the maximal fractional protection and [TRAP] is the concentration of TRAP. Values for observed rate constants (kobs) were obtained by fitting the data to equation (3):

(2)

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jmb.2004.10.071

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Edited by D. E. Draper (Received 31 May 2004; received in revised form 21 October 2004; accepted 24 October 2004)