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Structure, Vol. 12, 1269–1280, July, 2004, 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.str.2004.05.005 Crystal Structure of Activated HutP: An RNA Binding that Regulates Transcription of the hut in Bacillus subtilis

Thirumananseri Kumarevel,1,2 Zui Fujimoto,2 loop structure that functions as a terminator. A cis-act- Ponnuraj Karthe,3 Masanao Oda,1 ing regulatory sequence required for induction of ex- Hiroshi Mizuno,2 and Penmetcha K.R. Kumar1,* pression of the hut structural by histidine has 1Institute for Biological Resources and Functions been identified. It forms an antiterminator structure and National Institute of Advanced Industrial is located just upstream of the hut structural genes, Science and Technology (AIST) partially overlapping the downstream terminator (Wray Tsukuba Central 6 and Fisher, 1994; Oda et al., 2000). The HutP is a 16.2 Tsukuba, Ibaraki 305-8566 kDa protein consisting of 148 amino acid residues and Japan is reported to be essential for higher level of expression 2 Department of Biochemistry of the hut operon (Oda et al., 1988). Interestingly, the National Institute of Agrobiological Sciences HutP protein binds to the regulatory sequence on the Tsukuba, Ibaraki, 305-8602 hut mRNA only when activated by L-histidine. Several Japan lines of evidence have indicated that HutP regulates 3 Center of Biophysical Sciences & Engineering expression of the downstream genes of the hut operon 1025 18th Street South by an antitermination mechanism (Oda et al., 1992, 2000; University of Alabama at Birmingham Wray and Fisher, 1994). It obviously is of importance to Birmingham, Alabama 35294 determine the conformation of HutP in the presence of L-histidine; this conformation is presumably necessary for binding to and stabilizing the antiterminator structure Summary in hut mRNA. Similar that regulate by binding to cognate HutP is an L-histidine-activated RNA binding protein mRNAs have been described. The regulatory proteins that regulates the expression of the histidine utilization identified to date from E. coli and B. subtilis are TRAP, (hut) operon in Bacillus subtilis by binding to cis-acting PyrR, LacT, BglG, SacT, and GlpP. These regulate the regulatory sequences on the hut mRNA. The crystal trp, pyr, lac, and glp , respectively (Houman et structure of HutP complexed with an L-histidine ana- al., 1990; Aymerich and Stenmetz, 1992; Babitzke and log showed a novel fold; there are four antiparallel ␤ Yanofsky, 1993; Arnaud et al., 1996; Lu et al., 1996; strands in the central region of each monomer, with Alpert and Siebers, 1997; Glatz et al., 1996). Many of two ␣ helices each on the front and back. Two HutP these proteins require initial activation by their specific monomers form a dimer, and three dimers are ar- ligand before performing their function in antitermination ranged in crystallographic 3-fold symmetry to form a or termination with their target sequences. BglG is acti- hexamer. A histidine analog was located in between vated by ␤-glucosides, SacT and SacY by sucrose, the two monomers of HutP, with the imidazole group TRAP by tryptophan, PyrR by uridine (UMP or UTP), of L-histidine hydrogen bonded to Glu81. An activation LacT by lactose, and GlpP by glycerol-3-phosphate. The mechanism is proposed based on the identification of BglG, SacT, and SacY proteins indirectly regulate bgl key residues of HutP. The HutP binding region in hut and sac operons, respectively. These proteins are phos- mRNA was defined: it consists of three UAG trinucleo- phorylated in the presence of their respective sub- tide motifs separated by four spacer nucleotides. Resi- strates. In addition, other transcriptional antiterminator dues of HutP potentially important for RNA binding proteins such as amiR (Wilson et al., 1996) and nasR were identified. (Lin and Stewart, 1998) have been reported from other bacteria. To date, the crystal structures have been Introduction solved for the attenuator/antiterminator proteins PyrR (Tomchick et al., 1998), SacY (Manivel et al., 1997; Van The histidine utilization (hut) genes of Bacillus subtilis Tilbeurgh et al., 1997), LicT (Van Tilbeurgh et al., 2001), are responsible for the degradation of L-histidine and and TRAP (Antson et al., 1995). But protein-RNA com- its use as a carbon or nitrogen source. The hut genes plexes have been solved for only two activated proteins, are arranged in a single hut operon, consisting of a TRAP and LicT (Antson et al., 1999; Yang et al., 2002). positive regulatory , hutP, followed by five struc- The HutP sequence and its cognate RNA sequence tural genes, hutH, hutU, hutI, hutG, and hutM (Chasin are not related to any known antiterminator protein and and Magasanik, 1968; Kimmhi and Magasanik, 1970; RNA antiterminator (RAT), respectively. Hence, it is rea- Oda et al., 1988; Yoshida et al., 1995) (Figure 1A). Expres- sonable to assume that a knowledge of the three-dimen- sion of the operon is regulated by catabolite repression sional structure of the active HutP would provide a better and by L-histidine-induced transcription antitermination understanding of its mechanism of antitermination, as (Oda et al., 1988). The RNA nucleotide sequence located well as the unraveling of a novel motif binding to RNA. between the hutP coding region and the coding region Amino acid sequence comparisons and a search of the for the five structural genes is predicted to form a stem- database have revealed HutP proteins from only three bacteria, namely, Bacillus halodurans, Bacillus an- *Correspondence: [email protected] thracis, and Bacillus cereus, with about 60% sequence Structure 1270

Figure 1. hut operon of Bacillus subtilis and Allosteric Activation by L-Histidine for RNA Binding (A) Schematic representation of the hut operon showing the arrangement of the hutP, termina- tor/antiterminator, and structural genes. The nucleotide regions ϩ498 to ϩ571 and ϩ459 to ϩ537 are proposed to form terminator and antiterminator structures, respectively (for ad- ditional details, see Figure 5). (B) Filter binding assay showing the binding of HutP to RAT (ϩ459 to ϩ537; 79-mer) only in the presence of L-histidine (10 mM). (C) Concentration of L-histidine required for activation of HutP for RAT binding. Binding reactions were carried out in binding buffer where 160 nM of HutP protein was first incu- bated with varying concentrations of L-histi- dine (1–40 mM) in the presence of 20 nM of RAT (79-mer). The reaction mixture was incu- bated for 15 min, applied to the filter, and washed, and the amount of radioactivity was quantitated using the bio image analyzer BAS 2500 (Fuji Film).

similarity. However, structural information for these pro- presence of L-histidine (Figure 1B). Thus, L-histidine teins are remains obscure. allosterically appears to control HutP-RAT interactions This paper reports on the crystal structure of HutP by modifying the conformational state of HutP. Titrating complexed with an L-histidine analog, HutP-L-histidine with 1–40 mM of L-histidine and analyzing for RNA bind- ␤-naphthylamide (HBN), refined to 2.8 A˚ resolution. The ing with a filter binding assay (Figure 1C) showed that HutP consisted of four antiparallel ␤ strands in the cen- 10 mM L-histidine was required for complete activation tral region and two ␣ helices located on both the front of HutP. and back sides. The HutP formed a hexamer with individ- ual subunits arranged as dimers and related by 3-fold Screening for L-Histidine Analogs Activating HutP rotational symmetry. It had a novel protein fold for bind- Various analogs of L-histidine were screened using a ing to RNA. Based on mutational analysis of the L-histi- filter binding assay in the presence of its RAT (79- dine binding site, an allosteric activation mechanism by mer, ϩ459 to ϩ537) to determine the chemical groups of L-histidine is proposed involving the amino acid resi- L-histidine responsible for the activation of HutP protein. dues Glu81 and Arg88. Since X-ray and gel filtration Among the analogs tested, L-histidine ␤-naphthylamide analyses revealed that the HutP protein forms a hex- (HBN) and L-histidine benzylester showed higher affinity

-nM) over L-histidine (10-fold; Figure 2). L-histi 30ف amer, a search was made for any repeats of nucleotides (Kd in the above RAT region and found three repeated units dine methyl ester and L-␤-imidazole lactic acid showed nM), and urocanic 300ف of UAG motifs. A shorter RNA (ϩ496 to ϩ515 nt) compris- similar affinity as L-histidine (Kd ing three UAG motifs with four nucleotide regions in acid, histamine, and L-histidinamide showed only weak between them is sufficient for efficient binding to the activation (data not shown). D-histidine, imidazole-4- HutP in the presence of 10 mM of L-histidine. Further, acetic acid, L-histidinol, ␣-methyl-DL-histidine, 1-methyl biochemical analysis on the shorter RNA revealed that L-histidine, 3-methyl L-histidine, and 3-2(thienyl)-L ala- HutP appears to require three UAG trinucleotide motifs nine failed to show any activation (data not shown). within this region, and spacer nucleotides have no direct Even though the results point to the importance of the role in binding. Finally, based on the electrostatic poten- imidazole ring of L-histidine in the activation of HutP, tial and mutational analyses, we are also able to specu- imidazole itself failed to activate the HutP protein, sug- late on the potential residues of HutP that interact with gesting that the imidazole ring along with the backbone RAT. of L-histidine are essential for the activation. Based on the assays, both L-histidine ␤-naphthylamide (HBN) and Results and Discussion L-histidine benzylester were considered excellent can- didates for generating crystals of the HutP complex, L-histidine is known to induce expression of hut operon but having slightly higher activity, HBN was chosen to by interacting with HutP protein (Oda et al., 2000). HutP prepare crystals of the binary complex for investigating was found to bind to its terminator RNA only in the the active conformation of HutP that binds to RAT. Crystal Structure of Antiterminator Protein-HutP 1271

Figure 2. Screening for Analogs of L-Histidine that Activate HutP for RAT Binding Increasing concentrations of HutP (10–640 nM) were equilibrated with L-histidine analogs, either 5 or 10 mM, in binding buffer. Then 79-mer RAT was added to 20 nM concentration and incubated for about 15 min. The contents were filtered and washed, and the radioactivity was determined as described in Figure 1C. The concentrations of L-histidine analogs used were 10 mM for (A) and 5 mM for (B), whereas the HutP concentrations used for (A) and (B) were 20–320 nM and 10–640 nM, respectively.

Crystal Structure of HutP B. cereus (Ivanova et al., 2003) aligned, with secondary For crystal structure determination of HutP, a single- structures depicted over the sequences. Sequence point mutant (Val51Ile) with a higher affinity for hut mRNA comparisons show that the C-terminal amino acid resi- in the presence of L-histidine (our unpublished data) dues are more conserved than the N-terminal residues. was used. In crystallization trials, the mutant yielded There were two molecules of HutP per asymmetric unit reproducible and well ordered crystals that diffracted with an approximate dimension of 57 A˚ ϫ 43 A˚ ϫ 34 A˚ beyond 3.0 A˚ compared to wild-type HutP. Both mutant (Figure 3B). In molecule A, a 310 helix was found in be- and wild-type HutPs formed hexamers in solution, either tween the ␣4 and ␤2 strands, whereas the same region in the presence or absence of L-histidine (Oda et al., was disordered in molecule B. Molecules A and B were 2000). The HutP was cocrystallized in the presence of related by a noncrystallographic 2-fold axis perpendicu- HBN. All diffractions were carried out in the synchrotron lar to the plane of Figure 3B, with the bound HBN mole- beam line at the Photon Factory (PF), Tsukuba, Japan. cule located in between molecules A and B in the vicinity X-ray data were collected under cryogenic conditions of a 2-fold axis. Hydrophobic residues of the ␤4 strand (100 K). Data collection and refinement statistics are and surrounding residues may have contributed given in Table 1, together with further details of the X-ray strongly to dimer formation, in addition to some ionic analysis. The structure was solved by the multiwave- forces. The total solvent-accessible surface area buried A˚ 2 per monomer. The loop 1000ف length anomalous dispersion (MAD) method (Hendrick- upon dimerization was son, 1991) with the use of the L III edge of Hg. The regions L3 (A110–119) and L4 (A130–137) in molecule asymmetric unit of the crystal belonged to the cubic A and loop regions L1 (B61–69), L2 (B88–99), and L3 space group P213 with a ϭ b ϭ c ϭ 95.41 A˚ and contained (B109–119) in molecule B were disordered. The side two HutP molecules. The structure was refined to a chains of residues Arg98, Glu113, Ser114, Glu115, crystallographic R factor of 0.23 (Rfree ϭ 0.28) at 2.80 A˚ Ile133, and Lys134 in molecule A and those of Lys5, resolution. Lys49, Lys69, Leu96, Leu97, Glu113 (density not ob- HutP belonged to the ␣/␤ family of proteins, whose served for this residue), Ser114, Glu115, Glu117, and structure consists of four ␣ helices and four ␤ strands Lys134 in molecule B were disordered; hence, these arranged in the order of ␣-␣-␤-␣-␣-␤-␤-␤ in the primary residues were modeled with Ala. The first five residues structure, and four antiparallel ␤ strands forming a ␤ of the N-terminal region were not modeled because of sheet in the order of ␤1-␤2-␤3-␤4 with two ␣ helices weak electron densities. each in the front and back of the ␤ sheet. Figure 3A Three HutP dimers were symmetrically arranged shows the sequences of HutP from four closely related around a crystallographic 3-fold axis to form the quater- species: B. subtilis (Oda et al., 1988), B. halodurans nary structure of the hexamer (Figure 3C). The apparent (Takami et al., 2000), B. anthracis (Read et al., 2003), and molecular weight of HutP, as determined by gel filtration, Structure 1272

Table 1. Data Collection and Refinement Statistics of Mutant HutP Diffraction Data

Space group P213 Unit cell a ϭ b ϭ c ϭ 95.41 A˚ Number of protein 2 molecules per asymmetric unit Solvent content 45.2%

No. of

Reflections Completeness (%) I/␴(I) Rmerge Dataset Wavelength (A˚ ) Resolution (A˚ ) (tot./last shell) (tot./last shell) (tot./last shell) (tot./last shell) Hg1 1.007 3.0 42909/6047 100.0/100.0 11.0/3.70 0.045/0.207 Hg2 0.995 3.0 42884/6047 100.0/100.0 9.5/3.50 0.047/0.213 Hg3 0.950 3.0 28414/6030 99.7/98.5 9.50/2.90 0.051/0.258 Native data 0.978 2.8 52705/7379 100.0/100.0 7.9/3.2 0.050/0.239

Refinement and Model Statistics Resolution 20–2.80 A˚ Total number of reflections 7359 used in refinement Number of reflections used 6960 (94.4%) for working set

Rfactor 0.23 Number of reflections used 399 (5.4%)

for Rfree

Rfree 0.28 Rmsd bond angles (Њ) 1.42 Rmsd bond lengths (A˚ ) 0.008 Rmsd improper angles (Њ) 0.840 Model Number of nonhydrogen atoms 2143 Number of HBN atoms 21 Number of water molecules 84 Average B Factor (A˚ 2) Protein 75.65 HBN 86.12 Solvent 59.30

Ramachandran Statistics

Most favored regions 85.7% Allowed regions 13.9% Generously allowed regions 0.4% Disallowed regions 0.0%

Rmerge ϭ⌺hkl⌺j|Ij(hkl) ϪϽI(hkl)Ͼ|/⌺hkl ⌺jϽI(hkl)Ͼ, where Ij(hkl) and ϽI(hkl)Ͼ are the intensity of measurement j and the mean intensity for the reflection with indices hkl, respectively. Rfactor, free ϭ⌺hkl||Fcalc(hkl)| Ϫ |Fobs(hkl)||/⌺hkl|Fobs|, where the crystallographic Rfactor is calculated including and excluding reflections in the refinement. The free reflections constituted 5.4% of the total number of reflections. Rmsd, root mean square deviation; I/␴(I), ratio of mean intensity to a mean standard deviation of intensity.

suggests that it exists as a hexamer (Oda et al., 2000). (CE1) of His77 of molecule A in another dimer and was The hexameric structure was stabilized by hydrophobic probably the reason for the higher affinity in this contacts between the dimers, especially by residues complex. 68–80 of molecule A, which made many hydrophobic A search of the SCOP (Murzin et al., 1995) database contacts with the neighboring dimer. Residue Tyr69 of (1.61) did not reveal a fold similar to the HutP fold. A molecule A hydrogen bonded with Arg88 of neighboring structure similarity search of the PDB (Berman et al., molecule A. Similarly, residues 90–98 and 131–136 in 2002) using DALI (Holm and Sander, 1996) identified only molecule B made contacts with residues 73–84 and 46– fragments of unrelated proteins containing ␤ strands. A 53, respectively, in the neighboring molecule B. Thus, maximum Z score of 5.0 was obtained with a fragment the hexamerization of HutP may be attributed to multiple of anti-sigma f-factor protein (PDB ID 1L0O). This had points of contact being made between molecules A and only an 8% sequence identity and very little resem- B. The surface buried by this association resulted in a blance to the HutP strands (rmsd, 22%), so the overall A˚ 2 per dimer. structure of the HutP was unrelated to any structures 2500ف loss in accessible surface area of The reason for the higher affinity in the case of the available in the PDB, including known RNA binding do- mutant was probably due to favorable hydrophobic con- mains such as the ribonucleo protein (RNP) domain, the tact of Ile51 of the molecule with Pro132 of molecule B hnRNP K homology (KH) domain, the double-stranded of the neighboring dimer. The naphthalene moiety (C2) RNA binding domain, the arginine rich motif (ARM), and of HBN made hydrophobic contact with the side chain the oligonucleotide/oligosaccharide binding (OB) fold Crystal Structure of Antiterminator Protein-HutP 1273

Figure 3. Amino Acid Sequence Comparison of HutP and Structure of the Complex of HutP with HBN (A) Sequence alignment of HutP from B. subtilis (BS), B. halodurans (BH), B. anthracis (BA), and B. cereus (BC). Conserved residues are indicated by red letters. The secondary structural elements in the primary sequences of HutP are indicated as ␣ helices (bars), ␤ strands (arrows), and loops (lines). (B) Stereoview of the HutP dimer along the noncrystallographic 2-fold axis. HutP is shown in a ribbon diagram with labels for ␣ helices and ␤ strands (molecule A, green; molecule B, red) and HBN as a ball-and-stick model colored by atom type (nitrogen, blue; carbon, green; oxygen, red). The N-terminal and C-terminal of HutP are indicated by N and C, respectively. (C) Stereoview of HutP hexamer along the 3-fold axis with HBN. domain (Nagai et al., 1990; Musco et al., 1996; Bycroft cent molecules (Figures 4A and 4B). The strand ␤4of et al., 1995; Kharrat et al., 1995; Murzin, 1993; Legault molecule A and ␣4 of molecule B were responsible for et al., 1998). the interactions with L-histidine. The imidazole ring am- ide nitrogen ND1 was hydrogen bonded with the side L-Histidine Binding Site chain oxygen (OE2) of Glu81 of molecule B. The imida- The imidazole ring of L-histidine was buried in an hy- zole ring nitrogen (NE2) was hydrogen bonded to the drophobic pocket made up of residues from two adja- backbone carbonyl oxygen of Glu139 of molecule A and Structure 1274

Figure 4. Details of the Interactions of HutP-HBN Complex (A) Molecular surface representation of HutP interacting with HBN. The surfaces are colored according to the electrostatic potential (blue, positive; red, negative). The imidazole ring of HBN is buried within the hydrophobic pocket of the HutP. (B) A close stereoview of the HBN binding site in HutP. Hydrogen bonds are indicated by broken lines. HBN is represented by a ball-and- stick model.

made another hydrophobic contact with the phenyl ring Minimal RAT for HutP Binding (CD2) of Phe141 of molecule A. The ␣-amino nitrogen It has been suggested that hut mRNA forms two different of histidine was hydrogen bonded to the backbone car- secondary structures depending on the binding of the bonyl oxygen of Phe141 of molecule A. The backbone activated HutP (Oda et al., 2000) (Figure 5). The regions carbonyl nitrogen of Gly85 of molecule B made a hydro- that fold into alternative structures are shown within gen bond with the carboxy oxygen of L-histidine. The nucleotides ϩ459 to ϩ572. The free energies calculated backbone oxygen of the Glu81 residue made another using the RNA structure program version 3.71 (Mathews weak hydrogen bond with the nitrogen of the napthylam- et al., 1999) appear to show hut mRNA forming a stable ide group. Thus, the amino and carboxy moieties of the terminator structure in the absence of activated HutP HBN were bound in between the two molecules of HutP between nucleotides ϩ498 and 572 with a higher free through four hydrogen bonds: two of them with ␤ strand energy (⌬G ϭϪ31.5), whereas upon binding to activated residues of molecule A, two with helical residues of HutP, the region between ϩ459 and ϩ537 appears to molecule B and hydrophobic contacts with Phe141 of show hut mRNA forming a destabilized RNA structure molecule A. The other interaction observed was a salt (antiterminator) with less than half the free energy (⌬G ϭ bridge between Glu81 of molecule B and Arg88 of mole- Ϫ13.8) (Oda et al., 2000). The active HutP prevents tran- cule A. As a result of the extensive interactions between scription termination of the hut operon by binding to the the two molecules, the bound L-histidine imidazole terminator on mRNA, thereby stabilizing the antitermina- group was completely buried and inaccessible to the tor and inhibiting the formation of the downstream termi- solvent surface. The naphthylamide group (C5) also nator structure (Oda et al., 2000). At present, the minimal made an additional hydrophobic interaction with a histi- RAT sequence for HutP binding has been mapped in dine (His84 of molecule B) side chain. When the structure the region ϩ459 to ϩ537 nt of hut mRNA (79-mer); how- of HutP, cocrystallized with L-histidine benzylester and ever, the identity elements of RAT for binding have not L-histidine, was solved, the mode of binding was found been determined yet. Since X-ray and gel filtration analy- to be the same as in the case of HBN, and the HutP ses revealed that the HutP protein forms a hexamer structure was also similar to that in the HBN complex (trimers of dimers), a search was made for any repeats (data not shown). of nucleotides in the above RAT region. Interestingly, Crystal Structure of Antiterminator Protein-HutP 1275

Figure 5. Proposed Alternative Structures that Form within the hut mRNA Region Proposed alternative structures (terminator, ϩ498 to ϩ572; antiterminator, ϩ459 to ϩ537) that form within the hut mRNA region (ϩ459 to ϩ572). Nucleotides overlapping between the terminator and antiterminator regions (U498 to G537) are indicated by black letters. Structures are drawn with an RNA structure program (Mathews et al., 1999), and ⌬G values are derived from the same program. three UAG repeats with a four-nucleotide spacer in be- HutP. The results showed that the spacer region con- tween the triplets were found between ϩ498 and ϩ514 taining the four U residues was the most optimum for (17-mer), suggesting that this may be the HutP minimal efficient binding to HutP (Figure 6B). Any shortening of RNA binding element. Moreover, the data presented in the spacer region by 2 nt abolished the binding com- this paper suggest a modified secondary structure pletely, and a 3 nt spacer reduced the affinity over 1000- where the segment of nucleotides 498–514 is not form- fold. When the spacer sequence lengths were increased ing a hairpin but is bound to the HutP protein. to 5 and 6 nt, the RNA binding affinities to HutP were To test this possibility, a 20-mer RNA consisting of decreased by 25% and 50%, respectively. The results regions ϩ496 to ϩ515 of RAT was chemically synthe- suggest that the 20-mer RNA is the smallest binding sized and analyzed for binding with a filter binding assay element for HutP recognition, with each dimer in the using increasing concentrations of HutP in the presence hexamer of the activated HutP recognizing one unit of and absence of L-histidine. The binding studies revealed trinucleotide base (UAG). The 4 nt spacer length in be- that the HutP binds with similar affinity either to the 20- tween the trinucleotide (UAG) repeats is optimal in order mer RNA or the 79-mer RNA (ϩ459 to ϩ537) in the to reach the next binding site in the hexamer context; presence of L-histidine (Figure 6A). Binding data with however, when we reduced the spacer length, the UAG an additional synthetic RNA, in which four spacer nucle- motif failed to reach the next binding site, while increas- otides were substituted for four U nucleotides in the ing the spacer length diminishes binding, suggesting spacer region of RAT (20-mer) between the UAG tri- that the extra nucleotides could cause the formation of nucleotide repeats, showed that the spacer sequences a bulge, thereby allowing the spacing between the three have only a minimal role in HutP recognition, causing docking sites to be maintained. only a 2-fold increase in affinity for HutP, very likely Next, we carried out gel-shift assays to determine due to favorable stacking interactions with protein. In a the molecular interaction ratio for hexameric HutP and further test of the requirement of the spacer region, four cognate RNA (20-mer). By these analyses, initially, we 20-mer RNA molecules were constructed containing observed a single ternary complex band on the native spacer lengths of 2, 3, 5, and 6 poly “U” nucleotides gel (lanes 1 and 2 in Figure 6C). Based on this observa- and tested using the filter assay for binding to activated tion, we could not clearly suggest whether the observed Structure 1276

Figure 6. Identification of Important Regions in RAT and Gel-Shift Analysis (A) Identification of important bases and minimal RAT for HutP binding. The region of the 20-mer RNA is represented in bold letters in the 79-mer RNA. A filter binding assay was used for equilibrium dissociation analysis of the RNAs recognized by HutP. Binding reactions were carried out in binding buffer with 20 nM of RNAs in the presence of varying concentrations of HutP (20–320 nM) containing 10 mM of

L-histidine. The amount of complex retained on the filter after washing was used to calculate the equilibrium dissociation constants, Kd, using GraphPad Prism software version 3.0 (Graphpad Software). (B) Importance of spacer length between UAG motifs of RAT. Five RNAs that differ in spacer length from 2U to 6U residues were used for the analyses. Filter binding reactions were carried out in binding buffer containing 20 nM of different RNAs and varying concentrations of

HutP protein (10–1280 nM) in the presence of 10 mM of L-histidine. The contents were filtered and washed, and the Kd determined as mentioned above. (C) Gel-shift assay showing HutP-RAT complex. All binding reactions were carried out in binding buffer in the presence of 10 mM L-histidine. Lanes 1 and 2 are mixed with only labeled RNA (10 kcpm) with 1.25 and 2.50 ␮M of HutP, respectively. Lanes 3 to 8 represent 1.25 ␮Mof HutP with 20-mer RNA (10 kcpm) in the presence of increasing concentrations of unlabeled RNA as following: lane 3, 0.4 ␮M; lane 4, 0.8 ␮M; lane 5, 1.2 ␮M; lane 6, 1.6 ␮M; lane 7, 3.2 ␮M; lane 8, 6.4 ␮M. The position of free RNA is indicated by an arrowhead, and ternary complex 1:1 and 1:2 complexes are indicated by C1 and C2, respectively.

band corresponds to a 1:1 or 1:2 molar ratio of RAT-HutP Evaluation of the L-Histidine Binding Site complex. To clarify this issue, we carried out competitive by Mutagenic Analysis binding analysis in the presence of increasing concen- To evaluate the interactions with HBN, three mutants trations of cold 20-mer RAT under the same conditions (Glu81Ala, Phe141Ala, and Arg88Ala) were prepared, and found a second band (C2; Figure 6C, lanes 3–8) and their binding to RAT was analyzed by filter binding below the first band (C1) at a higher concentration of and gel-shift assays (Figures 7A and 7B). Before analyz- cold RNA. The observed C2 complex shift on the native ing the mutants, native gel electrophoresis was carried gel appeared to be reasonable: a loss of 6500 Da mass. out to confirm the formation of the hexamer (data not From these studies, it suggests that the HutP has the shown). The Glu81Ala mutant failed to activate the HutP, two potential binding sites for the cognate RNA. which was attributed to the loss of one hydrogen bond Crystal Structure of Antiterminator Protein-HutP 1277

the hydrogen bond network with the side chain of Glu81 and protein backbone atoms. This may lead to a rear- rangement of the local structures near the L-histidine binding site, such as one favoring the formation and stabilization of the ␤4 strand for RNA binding in the presence of the ligand. In order to obtain further details on the mechanism of activation of HutP by L-histidine, work is currently in progress to crystallize HutP alone (ligand-free form) for higher resolution studies, as well as to analyze the crystal structures of mutants of HutP near the L-histidine binding sites (Glu81Ala, Phe141Ala, and Arg88Ala).

Identification of Potential RNA Binding Sites of HutP To supplement our current findings, further mutational analyses were carried out to identify the functional groups within the UAG motif. Our recent studies re-

vealed that NH2 groups of the A and G bases were found to exert great influence in HutP-RAT binding (our unpublished data). Since protein-RNA interactions re- sponsible for binding in complexes are generally guided by the electrostatic potential, the electrostatic potential surface was calculated for the hexameric HutP using the GRASP program. The goal was to find potential

cognate sites that could accept the donor NH2 groups of the A and G bases from the UAG motifs (Figure 8A). It is noteworthy that residues Glu55 (indicated by an arrow in Figure 8A) and Glu137 (indicated by an arrow- Figure 7. Mutational Analysis of the L-Histidine Binding Site head in Figure 8A) of HutP are exposed to the solvent. (A) Equilibrium binding studies to analyze the binding ability of HutP Conceivably, they may be interacting with core elements and L-histidine binding site mutants to 20-mer RAT. of UAG. Further, the distance between these residues (B) Gel-shift assay of HutP mutants possessing modifications sur- correlates well with the distance between the groups of rounding the L-histidine binding site. Binding reactions were carried interest in the RNA. To decide on these potential sites, out with 40 nM of 20-mer RAT containing 10 mM L-histidine in the absence (lane 1) and presence of a 320 nM concentration of HutP the two residues were substituted with Ala, and their (lane 2), Glu81Ala (lane 3), Phe141Ala (lane 4), and Arg88Ala (lane 5). RNA binding was checked by gel-shift assay. Interest- ingly, Glu137Ala (lane 3 in Figure 8B) completely failed to bind to the RNA, whereas the Glu55Ala mutant (lane between the imidazole group of L-histidine and the side 2 in Figure 8B) did not affect the binding to RNA (Figure chain of Glu81 (lane 3, Figure 7B). With the Phe141Ala 8B). Based on this analysis, we presume that the NH2 mutant, the binding ability was reduced by 3-fold (Kd group of A or G interacts with residue Glu137 and in 687nM) (lane 4, Figure 7B). Substitution of Phe141 in doing so favors the ␤4 strand for the RNA binding uponف the other three bacterial HutPs with another hydropho- activation by L-histidine. bic residue (Ile) suggested that the hydrophobic contri- In summary, this paper describes the crystal structure bution is important. Substitution of Arg88 with Ala also of allosterically activated HutP protein and a novel pro- abolished binding to RAT (lane 5, Figure 7B), which was tein fold. The active structure of HutP forms a dimer attributed to the loss of a salt bridge between Glu81 of in an asymmetric unit, and three of these dimers are molecule B and Arg88 of molecule A. It is known that arranged in a 3-fold symmetrical fashion to form the residues Glu81 and Arg88 are well conserved in the hexamer. The bound L-histidine analog, HBN, is located other three bacterial HutP proteins. The gel-shift analy- in between molecules of the dimer. The mode of binding ses demonstrate which residues of HutP are important suggests that the imidazole ring, as well as the backbone for L-histidine binding to form the ternary complex. of L-histidine, is responsible for the activation of HutP. Since the ternary complex appeared to dissociate during The surrounding residues of ␣4 and ␤4 constitute a hy- electrophoresis, the gel-shift analyses were comple- drophobic pocket and facilitate the accommodation of mented with filter binding assays to calculate their bind- the ligand. Once the ligand enters into the pocket, it ing constants. expands the hydrogen bond network with side chains Taken together, the present results provide strong as well as the backbone of HutP. This may be a key evidence that a hydrophobic pocket exists that stabi- feature in the activation process, which is required to lizes the dimer interface by the formation of a salt bridge allow the binding of hut mRNA. Based on the formation between the monomers of HutP, Glu81 of molecule B, of a hexameric structure of HutP, we were able to define and Arg88 of molecule A. The hydrophobic pocket in the minimal binding region in the RAT. The binding analy- turn attracts the ligand (L-histidine analog) and expands sis suggests that each dimer of the hexamer can recog- Structure 1278

details of the antitermination mechanism and thus con- tributes to an understanding of the transcription regula- tion of the hut operon in Bacillus species.

Experimental Procedures

Overexpression and Purification of HutP The coding region of HutP containing a Val51Ile mutation was ampli- fied and cloned downstream to the T7 promoter site on pET5a (Promega), using appropriate restriction enzymes (data not shown). The single-point mutant Val51Ile originated spontaneously during our studies on hut operon in Bacillus subtilis and was found to have a higher affinity to hut mRNA than the wild-type HutP. The resultant plasmid (pETHP4) was transformed into E. coli BL21 (DE3), and the HutP was overexpressed at mid-log phase by the addition of IPTG (1 mM). Harvested cells were dissolved in lysis buffer (50 mM Tris- HCl [pH 7.5], 2 mM EDTA, 0.1 mM DTT, 2 mM PMSF, and 5% glycerol) and sonicated for 10 min. After centrifugation at 16,000 ϫ g for 10 min, the supernatant was subjected to stepwise (10%–70%) ammonium sulfate precipitation to remove low molecular weight contaminant proteins. The HutP protein was sedimented after 80% ammonium sulfate precipitation. The pellet was dissolved in Tris- HCl buffer (50 mM Tris-HCl [pH 7.5], 2 mM EDTA, 0.1 mM DTT,

10 mM MgCl2, and 5% glycerol) and dialyzed overnight against the buffer. The dialyzed sample was loaded onto a DEAE-Toyopearl column and washed and eluted using a linear gradient of 0–400 mM NaCl in the same buffer. HutP-containing fractions (determined by SDS-PAGE) were pooled and dialyzed in phosphate buffer (10 mM

Na2HPO4 [pH 6.8], 2 mM EDTA, 10 mM MgCl2, and 5% glycerol). The HutP protein formed a precipitate under these conditions. The precipitated HutP was recovered by centrifugation and dialyzed against Tris-HCl buffer (50 mM Tris-HCl [pH 7.5], 2 mM EDTA, 0.1 mM

DTT, and 1 mM MgCl2) to solubilize the protein. The precipitation was repeated twice, which improved the purity to over 99%, after which the protein was concentrated to 10 mg/ml in the Tris-HCl buffer containing 20% glycerol for crystallization studies.

Crystallization and Data Collection Initial crystals of HutP were produced at 20ЊC by the hanging drop vapor diffusion method (McPherson, 1990) by adding 4 ␮l of protein solution to 4 ␮l of well solution (100 mM Na cacodylate buffer [pH 6.8], 0.5 M KBr, 30% polyethylene glycol [PEG] 2000 mono methyl Figure 8. Potential RNA Binding Sites of HutP ether) and adding 1 ␮l of 25 mM L-histidine ␤-naphthylamide (HBN). (A) Molecular surface of hexameric HutP colored according to elec- Crystals of the binary complex grew within a week. To improve the trostatic potential. The electrostatic potential was calculated and crystal size, the crystals were seeded into preequilibrated drops, visualized using GRASP. Basic regions are shown in blue and acidic resulting in large size crystals of up to 0.5 ϫ 0.3 ϫ 0.5 mm3 in in red. The electronegative potential for the Glu55 residue is indi- dimension. Crystals were soaked into mercury derivative Hg (CN)2 cated by arrows and for Glu137 by arrowheads. solution, and the data were collected at cryo-temperatures using (B) Mutational analysis of potential RNA binding site residues by a CCD (ADSC) detector. A complete multiwavelength anomalous gel-shift assay. Binding reactions were carried out with 40 nM of dispersion (MAD) data set at 100 K on an 18B beamline and a 20-mer RAT containing 10 mM of L-histidine in the absence (lane native data set at 6A beamline were collected at the Photon Factory,

1) and presence of 320 nM HutP mutants (lane 2, Glu55Ala; lane 3, Tsukuba, Japan. Crystals belonged to the cubic space group P213, Glu137Ala). with cell dimensions a ϭ b ϭ c ϭ 95.41 A˚ . The MAD and native data were processed up to 3.0 A˚ and 2.8 A˚ , respectively, using the MOSFLM suite (Leslie, 1990) (Table 1). nize one unit of trinucleotide (UAG) repeat, and 4 nt is required as a spacer between each of these triplets to Structure Determination and Refinement enable optimal binding at all three UAG binding sites. The binary complex structure of the HutP (HutP-HBN) was deter- The recognition motif, UAG trinucleotide repeats, is lo- mined using phasing information from MAD experiments. The initial cated just upstream to the terminator region of the RAT. positions of the three Hg sites were determined by SOLVE (Terwil- liger and Berendzen, 1999), and improvements were made of the The data presented here also suggest a modified sec- initial phases by density modification and phase extension from 3.0 ondary structure where the segment of nucleotides 498– to 2.8 A˚ using the program DM (CCP4, 1994). An interpretable elec- 514 is not forming a hairpin but is bound to the HutP tron density map was calculated and the initial atomic model was protein. Electrostatic potential analysis of the hexameric built on the basis of this density map using Quanta X-Build program HutP protein allowed us to identify one of the potential (Oldfield, 2001) and then refined by CNS (Brunger et al., 1998). The final structure was refined to a crystallographic R factor of 0.23 RNA binding site residues as Glu137, which is located ϭ ˚ ␤ (Rfree 0.28) at 2.80 A resolution, using synchrotron radiation X-ray in the 4 strand. While knowledge of the ternary complex data collected at cryo-temperatures (see Table 1). Figures were structure is essential to an unequivocal identification prepared using the programs Ribbons (Carson, 1997), Molscript of the key interactions proposed above, it also reveals (Kraulis, 1991), and Grasp (Nicholls et al., 1991). Crystal Structure of Antiterminator Protein-HutP 1279

RNA Synthesis Gel-Shift Assay The 79-mer RNA was enzymatically synthesized by in vitro transcrip- To evaluate the protein-RNA interaction ratio in solution, a gel-shift tion using T7 RNA polymerase on a synthetic DNA template. The assay was used. The 20-mer RNA was labeled at the 5Ј-end as template DNA containing the region ϩ 459 to ϩ536 of RAT was described above. The purified HutP was incubated in binding buffer chemically synthesized using a Model 394 RNA/DNA synthesizer (15 mM HEPES [pH 7.5], 30 mM NaCl, 5 mM MgCl2, and 10 ␮gof (Applied Biosystems). To generate the double-stranded DNA and yeast tRNA) at predetermined concentrations, and the RNA (20-mer) to add the T7 promoter to the template, two additional sets of probe was mixed at various concentrations in the presence of 10 primers were synthesized (forward primer: 5Ј-AGTAATACGACT mM L-histidine. The resulting reaction mixture was incubated for CACTATAGGGACATTCGGCGTTGGAATTAATCA-3Ј; and reverse 15 min at room temperature and mixed with 2 ␮l of 90% glycerol primer: 5Ј-CTTCTGTTTTCACGCATAGCCCCCTAT-3Ј). Using the before loading onto the gel. The free RNA and HutP-RNA complexes template DNA and primers, dsDNA template was prepared using a were resolved using 10% polyacrylamide gel (running buffer, 0.5ϫ PCR kit (ExTaq kit, Takara, Japan). The reaction mixture was cycled TBE; constant voltage, 15 mA; temperature, 4ЊC). The resulting auto- at 94ЊC for 1.15 min, 50ЊC for 1.0 min, and 72ЊC for 1.15 min for 10 radiograms were analyzed with the bioimage analyzer BAS 2500 cycles. The resulting PCR product was precipitated with ethanol, (Fuji Film). and about 2 ␮g was used for the in vitro T7 transcription in the presence of ␣-32P-CTP (Amersham). The in vitro transcription was Site-Directed Mutagenesis of HutP carried out at 37ЊC for 3 hr using the Ampliscribe transcription kit Initially, the HutP coding region was amplified by PCR from pETHP4 (Epicenter Technology). After transcription was completed, the and subcloned into the vector pET19b. Using the site-directed muta- products were treated with 2 units of DNase I (RNase free) for 10 genesis kit QuickChange (Stratagene), the HutP mutants (Glu55Ala, min at 37ЊC, to remove the template DNA, and mixed with an equal Glu81Ala, Arg88Ala, and Phe141Ala, Glu137Ala) were prepared. The volume of 2ϫ urea buffer (7 M urea, 50 mM EDTA, 90 mM Tris- proteins were expressed as His-tag fusion proteins in E. coli BL21 borate containing 0.05% bromophenol blue). The reaction mixtures (DE3) and purified using a Ni-NTA column (Qiagen). The His-tag was were denatured at 90ЊC for 2 min and fractionated on a 12% acryl- removed by digestion with enterokinase (EK; Invitrogen), and the amide gel containing 7 M urea. The RNAs were isolated from the HutP proteins were recovered. The resultant mutant HutP proteins gel, recovered by ethanol precipitation, and quantitated using ab- had one extra amino acid (His) at the N-terminal end. sorbance at 260 nm. The minimal RAT binding elements (20-mer RNA and its vari- Acknowledgments ants—20-mer RNA: 5Ј-CAUAGAUCUUAGACGAUAGG-3Ј; 20-mer Ј Ј spacer Subt.: 2U residues, 5 -CAUAG(U)2UAG(U)2UAGA-3 ; 3U resi- We thank Drs. N. Sakabe, M. Suzuki, and N. Igarashi for their kind Ј Ј Ј dues, 5 -CAUA G(U)3UAG(U)3UAGA-3 ; 4U residues, 5 -CAUAG(U)4 assistance shown at the Photon Factory, Tsukuba, Japan. One of the Ј Ј Ј UAG(U)4UAGA-3 ; 5U residues, 5 -CAUAG(U)5UAG(U)5UAGA-3 ; and authors (T.S.K.) is grateful to the Science and Technology Agency of Ј Ј 6U residues, 5 -CAUAG(U)6UAG(U)6UAGA-3 ) were chemically syn- Japan for a STA fellowship. This work was supported by funds from thesized using a Model 394 RNA/DNA synthesizer (Applied Biosys- METI and ORCS to P.K.R.K. and H.M., respectively. We thank Dr. tems) using phosphoramidite chemistry. All amidites were pur- Rick Conrad, Prof. E. Westhof, Dr. S. Nishikawa, and Prof. O. Nureki chased from Glen Corporation. The functional groups of the for critical reading of the manuscript. synthesized RNAs were deprotected by established protocols (ABI Manual), and the RNA was purified on 15% acrylamide gel con- Received: February 5, 2004 taining 7 M urea. The RNAs at the 5Ј end were labeled with [␥-32P]ATP Revised: May 6, 2004 (Amersham) in the presence of T4 polynucleotide kinase (Takara, Accepted: May 10, 2004 Japan), and the labeled products were recovered after fractionation Published: July 13, 2004 on 15% PAGE. References Filter Binding Assay For evaluation of the binding activities of prepared RNAs (79-mer Alpert, C.A., and Siebers, U. (1997). The lac operon of Lactobacillus RNA, 20-mer RNA and its variants), as well as for efficient screening casei contains lacT, a gene coding for a protein of the Bg1G family of L-histidine analogs, a filter binding assay was used as described of transcriptional antiterminators. J. Bacteriol. 179, 1555–1562. below. The purified HutP was incubated in binding buffer (15 mM Antson, A.A., Otridge, J., Brzozowski, A.M., Dodson, E.J., Dodson, HEPES [pH 7.5], 30 mM NaCl, 5 mM MgCl ,10␮g of yeast tRNA) 2 G.G., Wilson, K.S., Smith, T.M., Yang, M., Kurecki, T., and Gollnick, P. at various concentrations to give final concentrations of 10–640 nM, (1995). The structure of trp RNA-binding attenuation protein. Nature and L-histidine or its analogs were added to a final concentrations 374, 693–700. of 10 mM. For calculating HutP concentrations, 97.2 kDa was used as the molecular mass of hexameric HutP. To this reaction mixture, Antson, A.A., Dodson, E.J., Dodson, G., Greaves, R.B., Chen, X.-P., RNA was added to a final concentration of 20 nM. The RNA was and Gollnick, P. (1999). Structure of the trp RNA-binding attenuation previously denatured and folded in binding buffer at room tempera- protein, TRAP, bound to RNA. Nature 401, 235–242. ture. The reaction mixture was incubated for 15 min at 27ЊC and Arnaud, M.D., Debarbouille, M., Rapoport, G., Saier, M.H., Jr., and applied to a Millipore nitrocellulose membrane (HAWP filter, 0.45 Reizer, J. (1996). In vitro reconstitution of transcriptional attenuation ␮M, 13.0 mm diameter) previously equilibrated in the binding buffer. by the SacT and SacY proteins of Bacillus subtilis. J. Biol. Chem. The membrane was washed once with 1 ml of binding buffer con- 271, 18966–18972. taining L-histidine or its analogs. In the control experiments, the Aymerich, S., and Stenmetz, M. (1992). Specificity determinants and L-histidine or its analogs were not included in the reaction mixture, structural features in the RNA target of the bacterial antiterminator and washing was omitted. In the analog-searching studies for acti- proteins of the BgiG/SacY family. Proc. Natl. Acad. Sci. USA 89, vation of HutP, all the above conditions were the same, except lower 10410–10414. concentrations of HutP (20–400 nM) and L-histidine analogs (either Babitzke, P., and Yanofsky, C. (1993). Reconstitution of Bacillus 10 or 5 mM) were used in both binding and washing buffer. In the subtilis trp attenuation in vitro with TRAP, the trp RNA-binding atten- L-histidine titration studies carried out to determine the concentra- uation protein. Proc. Natl. Acad. Sci. USA 90, 133–137. tion required for activation, the HutP was incubated with various concentrations (1–40 mM) of L-histidine, and the binding assay was Berman, H.M., Battistuz, T., Bhat, T.N., Bluhm, W.F., Bourne, P.E., carried out with 20 nM of RAT (79-mer). The amount of RNA (radioac- Burkhardt, K., Feng, Z., Gilliland, G.L., Iype, L., Jain, S., et al. (2002). tivity) retained on the filter was quantitated using bioimage analyzer The Protein Data Bank. Acta Crystallogr. D. Biol. Crystallogr. 58, 899–907. BAS 2500 (Fuji Film). The equilibrium dissociation constants (Kd) were determined for different RNA-protein complexes using a Brunger, A., Adams, P.D., Clore, G.M., Gros, P., Grosse-Kuntsleve, GraphPad Prism 3.0 nonlinear regression algorithm (GraphPad R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., and Read, Software). R.J. (1998). Crystallography and NMR system: a new software sys- Structure 1280

tem for macromolecular structure determination. Acta Crystallogr. and stability of the KH domain: molecular insights into the fragile X D. Biol. Crystallogr. 54, 905–921. syndrome. Cell 85, 237–245. Bycroft, M., Grunert, S., Murzin, A.G., Proctor, M., and St Johnston, Nagai, K., Outbridge, C., Jessen, T.H., Li, J., and Evans, P.R. (1990). D. (1995). NMR solution structure of a dsRNA binding domain from Crystal structure of the RNA-binding domain of the U1 small nuclear Drosophila staufen protein reveals homology to the N-terminal do- ribonucleoprotein A. Nature 348, 515–520. main of ribosomal protein S5. EMBO J. 14, 3563–3571. Nicholls, A., Sharp, K.A., and Honig, B. (1991). Protein folding and Carson, M. (1997). Ribbons. Methods Enzymol. 277, 493–505. association: insights from the interfacial and thermodynamic prop- erties of hydrocarbons. Proteins 11, 281–296. CCP4 (Collaborative Computing Project 4) (1994). The CCP4 Suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crys- Oda, M., Sugishita, A., and Furukawa, K. (1988). Cloning and nucleo- tallogr. 50, 760–763. tide sequences of histidase and regulatory genes in the Bacillus subtilis hut operon and positive regulation of the operon. J. Bacteriol. Chasin, L.A., and Magasanik, B. (1968). Induction and repression of 170, 3199–3205. the histidine-degrading enzymes of Bacillus subtilis. J. Biol. Chem. Oda, M., Katagai, T., Tomura, D., Shoun, H., Hoshino, T., and Furu- 243, 5165–5178. kawa, K. (1992). Analysis of the transcriptional activity of the hut Glatz, E., Nilsson, R.P., Rutberg, L., and Rutberg, B. (1996). A dual promoter in Bacillus subtilis and identification of a cis-acting regula- role for the Bacillus subtilis glpD leader and the GlpP protein in the tory region associated with catabolite repression dowmstream from regulated expression of glpD: antitermination and control of mRNA the site of transcription. Mol. Microbiol. 6, 2573–2582. stability. Mol. Microbiol. 19, 319–328. Oda, M., Kobayashi, N., Ito, A., Kurusu, Y., and Taira, K. (2000). Cis- Hendrickson, W.A. (1991). Determination of macromolecular struc- acting regulatory sequences for antitermination in the transcript of tures from anomalous diffraction of synchrotron radiation. Science the Bacillus subtilis hut operon and histidine-dependent binding of 254, 51–58. HutP to the transcript containing the regulatory sequences. Mol. Holm, L., and Sander, C. (1996). A review of the use of protein Microbiol. 35, 1244–1254. structure comparison in protein classification and function identifi- Oldfield, T.J. (2001). A number of real-space torsion-angle refine- cation. Science 273, 595–602. ment techniques for proteins, nucleic acids, ligands and solvent. Acta Crystallogr. D Biol. Crystallogr. 57, 82–94. Houman, F., Diaz-Torres, M.R., and Wright, A. (1990). Transcriptional antitermination in the bgl operon of E. coli is modulated by a specific Read, T.D., Peterson, S.N., Tourasse, N., Baillie, L.W., Paulsen, I.T., RNA binding protein. Cell 62, 1153–1163. Nelson, K.E., Tettelin, H., Fouts, D.E., Eisen, J.A., et al. (2003). The genome sequence of Ames and comparison to Ivanova, N., Sorokin, A., Anderson, I., Galleron, N., Candelon, B., closely related bacteria. Nature 423, 81–86. Kapatral, V., Bhattacharyya, A., Reznik, G., Mikhailova, N., Lapidus, A., et al. (2003). Genome sequence of Bacillus cereus and compara- Takami, H., Nakasone, K., Takaki, Y., Maeno, G., Sasaki, R., Masui, N., Fuji, F., Hirama, C., Nakamura, Y., Ogasawara, N., et al. (2000). tive analysis with Bacillus anthracis. Nature 423, 87–91. Complete genome sequence of the alkaliphilic bacterium Bacillus Kharrat, A., Macias, M.J., Gibson, T.J., Nilges, M., and Pastore, A. halodurans and genomic sequence comparison with Bacillus subti- (1995). Structure of the dsRNA binding domain of E. coli RNAse III. lis. Nucleic Acids Res. 28, 4317–4331. EMBO J. 14, 3572–3584. Terwilliger, T.C., and Berendzen, J. (1999). Automated MAD and MIR Kimmhi, Y., and Magasanik, B. (1970). Genetic basis of histidine structure solution. Acta Crystallogr. D. Biol. Crystallogr. 55, 849–861. degradation in Bacillus subtilis. J. Biol. Chem. 245, 3545–3548. Tomchick, D.R., Turner, R.J., Switzer, R.L., and Smith, J.L. (1998). Kraulis, P.J. (1991). MOLSCRIPT: a program to produce both de- Adaptation of an enzyme to regulatory function: structure of Bacillus tailed and schematic plots of protein structures. J. Appl. Crystallogr. subtilis PyrR, a pyr RNA-binding attenuation protein and uracil phos- 24, 946–950. phoribosyltransferase. Structure 6, 337–350. Legault, P., Li, J., Mogridge, J., Kay, L.E., and Greenblatt, J. (1998). Van Tilbeurgh, H., Manivel, X., Aymerich, S., Lhoste, J.M., Dumas, NMR structure of the bacteriophage lambda N peptide/boxB RNA C., and Kochoyan, M. (1997). Crystal structure of a new RNA-binding complex: recognition of a GNRA fold by an arginine-rich motif. Cell domain from the antiterminator protein SacY of Bacillus subtilis. 93, 289–299. EMBO J. 16, 5030–5036. Leslie, A.G.W. (1990). Crystallographic Computing (Cambridge, UK: Van Tilbeurgh, H., Le Coq, D., and Declerck, N. (2001). Crystal struc- Oxford University Press). ture of an activated form of the PTS regulation domain from the LicT transcriptional antiterminator. EMBO J. 20, 3789–3799. Lin, J.T., and Stewart, V. (1998). Nitrate assimilation by bacteria. Wilson, S.A., Wachira, S.J., Norman, R.A., Pearl, L.H., and Drew, R.E. Adv. Microb. Physiol. 39, 1–30. (1996). Transcription antitermination regulation of the Pseudomonas Lu, Y., Turner, R.J., and Switzer, R.L. (1996). Function of RNA sec- aeruginosa amidase operon. EMBO J. 15, 5907–5916. ondary structures in transcriptional attenuation of the Bacillus subti- Wray, L.V., Jr., and Fisher, S.H. (1994). Analysis of Bacillus subtilis lis pyr operon. Proc. Natl. Acad. Sci. USA 93, 14462–14467. hut operon expression indicates that histidine-dependent induction Manivel, X., Yang, Y., Strub, M.P., Kochoyan, M., Steinmetz, M., and is mediated primarily by transcriptional antitermination and that Aymerich, S. (1997). From genetic to structural characterization of amino acid repression is mediated by two mechanisms: regulation a new class of RNA-binding domain within the SacY/BglG family of of transcription initiation and inhibition of histidine transport. J. Bac- antiterminator proteins. EMBO J. 16, 5019–5029. teriol. 176, 5466–5473. Mathews, D.H., Sabina, J., Zuker, M., and Turner, D.H. (1999). Ex- Yang, Y., Declerck, N., Manivel, X., Aymerich, S., and Kochoyan, M. panded sequence dependence of thermodynamic parameters im- (2002). Solution structure of the LicT-RNA antitermination complex: proves prediction of RNA secondary structure. J. Mol. Biol. 288, CAT clamping RAT. EMBO J. 21, 1987–1997. 911–940. Yoshida, K., Sano, H., Seki, S., Oda, M., Fujimura, M., and Fujita, Y. McPherson, A. (1990). Current approaches to macromolecular crys- (1995). Cloning and sequencing of a 29 kb region of the Bacillus tallization. Eur. J. Biochem. 189, 1–23. subtilis genome containing the hut and wapA loci. Microbiol. 141, 337–343. Murzin, A.G. (1993). OB (oligonucleotide/oligosaccharide binding)- fold: common structural and functional solution for nonhomologous sequences. EMBO J. 12, 861–867. Accession Numbers

Murzin, A.G., Brenner, S.E., Hubbard, T., and Chothia, C. (1995). The coordinates and structure factors for the HutP complex with SCOP: a structural classification of proteins database for the investi- L-histidine analog have been deposited in the Protein Data Bank gation of sequences and structures. J. Mol. Biol. 247, 536–540. under the accession code 1VEA. Musco, G., Stier, G., Joseph, C., Castiglione Morelli, M.A., Nilges, M., Gibson, T.J., and Pastore, A. (1996). Three-dimensional structure