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Structural basis of HutP-mediated transcription anti-termination
Structural basis of HutP-mediated transcription anti-termination Penmetcha KR Kumar1, Thirumananseri Kumarevel1,* and Hiroshi Mizuno2 Bacteria often use anti-terminator proteins to sense a specific metabolite signal and direct RNA polymerase to either terminate transcription or transcribe the downstream genes of an operon. Although many proteins that regulate various operons using this mechanism have been identified, insights into their activation processes before cognate mRNA binding have remained obscure. HutP from Bacillus subtilis regulates the hut operon by an anti-termination mechanism. Recently, several crystal structures of HutP [apo-HutP, HutP–L-histidine (and histidine analog), HutP–L-histidine–Mg2+ and HutP–Lhistidine–Mg2+–RNA] have been reported. These structural and functional studies of HutP have revealed how the protein undergoes conformational changes in response to two key components: L-histidine and Mg2+ ions. Addresses 1 Functional Nucleic Acids Group, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1 Higashi, Tsukuba City 305-8566, Japan *Current address: Structural and Molecular Biology Laboratory, RIKEN Harima Institute at Spring-8. 1-1-1 Kouto, Sayo-cho, Hyogo 679-5148, Japan 2 NEC Soft Ltd, 1-18-7, Shinkiba, Koto-ku, Tokyo 136-8627, Japan Corresponding author: Kumar, Penmetcha KR ([email protected])
Current Opinion in Structural Biology 2006, 16:18–26 This review comes from a themed issue on Protein–nucleic acid interactions Edited by Gregory D Van Duyne and Wei Yang Available online 19th January 2006 0959-440X/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2006.01.005
Introduction Anti-terminator proteins control gene expression by recognizing control signals within cognate transcripts and preventing transcription termination. Examples of such regulatory proteins in Escherichia coli and Bacillus subtilis include BglG, SacT, SacY, TRAP and PyrR, which are known to regulate the bgl, sac, trp and pyr operons, respectively [1–5]. Many of these proteins exert their function as anti-terminators or attenuators upon binding to target sequences within nascent mRNAs. Another such protein is HutP, an anti-terminator protein that regulates the histidine utilization (hut) operon in B. subtilis [6,7]. The hut operon consists of five histidine utilization genes, hutH, hutU, hutI, hutG and hutM, which encode histidase, Current Opinion in Structural Biology 2006, 16:18–26
urocanase, imidazolone propionate amino hydrolase, formiminoglutamate hydrolase and histidine permease, respectively, and a positive regulatory gene, hutP (Figure 1a) [8–11]. The hutP gene is located just downstream of the hut promoter, whereas the histidine utilization genes are located far downstream of the promoter [10]. Expression of the operon is regulated by catabolite repression and by L-histidine-induced transcription antitermination. The mRNA transcript nucleotide sequence located between the hutP coding region and the coding region for the five histidine utilization genes is predicted to form a stem-loop structure that functions as a terminator (Figure 1b). In addition, a cis-acting regulatory sequence required for histidine-mediated induction of hut structural gene expression has been identified. It forms an anti-terminator structure and is located just upstream of the hut histidine utilization genes, partially overlapping the downstream terminator [7,12]. HutP is a 16.2 kDa protein consisting of 148 amino acid residues. HutP also exists in three other Bacillus species, including B. anthracis, B. cereus and B. halodurans, with 60% sequence identity. Sequence comparison of the Bacillus HutP proteins revealed that the C-terminal amino acid residues are more conserved than the Nterminal residues. Interestingly, the HutP protein binds to the terminator region within the hut mRNA and enhances hut structural gene expression only when activated by L-histidine. Several lines of evidence have indicated that HutP regulates the expression of the downstream genes of the hut operon by an anti-termination mechanism [6,7,12]. It is important to determine the conformational changes within HutP that allow it to recognize cognate sequences within the hut mRNA. Although other attenuator or anti-terminator proteins, such as TRAP [13,14] and LicT [15,16], form binary and ternary complexes, the conformational changes that the proteins undergo within these structures before binding to their cognate RNAs have remained obscure. Recently, the conformational changes that occur in HutP before cognate RNA binding have been described. Furthermore, these studies proposed how the terminator of the hut operon is destabilized by the activated HutP, facilitating the synthesis of the full-length transcript by RNA polymerase. The conformational changes induced by L-histidine and Mg2+ are of great interest, and are described in this review.
Structure of the HutP–L-histidine analog complex The first reported crystal structure of HutP was the HutP–L-histidine b-naphthylamide (L-histidine analog) www.sciencedirect.com
HutP-mediated transcription anti-termination Kumar, Kumarevel and Mizuno 19
Figure 1
The hut operon of B. subtilis. (a) Schematic representation of the hut operon, showing the arrangement of the hutP gene, terminator/anti-terminator region and histidine utilization genes. (b) The proposed hut mRNA terminator structure. Within the HutP-recognition motif, the UAG triplets are shown in pink. The stop codon of the hutP gene and the start codon of the hutH gene are shown in green.
complex structure, referred to in this review as the HutP– L-histidine analog complex [17,18 ]. In this complex, the HutP protein adopts an a/b structure, consisting of four a helices and four b strands. These are arranged in the order a-a-b-a-a-b-b-b in the primary structure; the four antiparallel b strands form a b sheet in the order b1b2-b3-b4, with two a helices each at the front and back of the b sheet. Three HutP dimers (molecules A and B related by non-crystallographic twofold symmetry) are related by threefold symmetry to form a hexamer, which seems to be the functional form [18] (Figure 2a,b). The apparent molecular weight of HutP, as determined by native gel studies, suggested that it exists as a hexamer [12]. The hexameric structure is stabilized by hydrophobic contacts between the dimers, especially residues 68– 80 of molecule A, which form many hydrophobic contacts with the neighboring dimer. Similarly, residues 90–98 and 131–136 of molecule B contact residues 73–84 and 46–53, respectively, of neighboring molecule B. The overall structure of HutP is unrelated to any structures currently available in the PDB, including known RNA-binding proteins, and thus it represents a novel RNA-binding motif. www.sciencedirect.com
HutP binds to its cognate terminator RNA only in the presence of L-histidine; it requires about 10 mM of Lhistidine for complete activation [12,18]. Therefore, it is believed that L-histidine allosterically controls HutP–hut mRNA interactions by modifying the conformation of HutP. The chemical groups of L-histidine responsible for activating the HutP protein have been determined by binding studies with various analogs of L-histidine [19]. Among these, the best analogs that supported HutP activation and binding to the hut mRNA were found to be L-histidine, L-histidine benzyl ester and L-histidine bnaphthylamide. These studies led to the conclusion that the imidazole group and the backbone of L-histidine are essential for the HutP activation process. To determine the details of the interaction with HutP, crystal structures of the HutP–L-histidine, HutP–L-histidine benzyl ester and HutP–L-histidine b-naphthylamide complexes have been solved [18]. These studies revealed that a hydrophobic pocket stabilizes the dimer interface through the formation of a salt bridge between the HutP monomers, involving Glu81 of molecule B and Arg88 of molecule A. L-Histidine binds within this hydrophobic pocket. The importance of these residues for mediating the interaction Current Opinion in Structural Biology 2006, 16:18–26
20 Protein–nucleic acid interactions
Figure 2
Structures of HutP complexes. (a) HutP hexamer viewed along the threefold axis, with the L-histidine analog (L-histidine b-naphthylamide, HBN) shown in pink as a ball-and-stick model. (b) HutP dimer viewed along the non-crystallographic twofold axis. HutP is shown as a ribbon diagram, with labeled a helices, b strands and loops (molecule A blue, molecule B green). (c) Dimer of apo-HutP. (d) Dimer of the ternary complex (HutP–L-histidine–Mg2+ ion). The bound L-histidine and Mg2+ ion are shown as a ball-and-stick model and a yellow sphere, respectively.
with a specific sequence within the hut mRNA, through their interaction with the L-histidine analog, was confirmed by site-specific mutational studies [18]. Recently, we have also solved two additional crystal structures, apo-HutP (Figure 2c) and the HutP–L-histidine–Mg2+ ternary complex (Figure 2d), to further understand the conformational changes that HutP is believed to undergo before binding to its cognate target site within hut mRNA [20]; a description of these structures will follow.
The minimal RNA motif and important chemical groups Initially, a minimal RNA region sufficient for efficient HutP binding was mapped to the region encompassing nucleotides +459 to +537 (79-mer RNA) of the hut mRNA [12]. As HutP exists as a hexamer, as mentioned above, a search for repeated sequences within the mapped region was made. Interestingly, three UAG repeats separated by Current Opinion in Structural Biology 2006, 16:18–26
four-nucleotide spacers were found. A short oligomer (spanning +496 to +515) was prepared, which bound as efficiently as the 79-mer RNA. Next, to address the importance of the spacer region to HutP binding, the spacer nucleotides were substituted by U bases, which revealed that the chemical groups of the spacer bases are not important, but that the presence of four nucleotides in the spacer is optimal for positioning the next binding site in the context of the hexamer [18]. To determine the RNA chemical groups that are essential for the interaction with HutP, an in vitro selection strategy and analyses of site-specific base substitutions were carried out [21]. These studies suggested that each HutP molecule recognizes one UAG motif; the first base (U) can be substituted by other bases, whereas the second and third bases (A and G) are essential for the interaction (NAG motif). Analyses of the chemical groups of the A and G bases within the UAG motif, using modified base analogs, suggested the importance of the exocyclic NH2 groups. In addition, in www.sciencedirect.com
HutP-mediated transcription anti-termination Kumar, Kumarevel and Mizuno 21
this motif, only the 20 -OH group of the A base is important for HutP recognition [18].
Divalent metal ion requirement All of the above studies showed that L-histidine is required for HutP to recognize the cognate target sequence within the hut mRNA. However, these studies were performed in a binding buffer containing 10 mM MgCl2. When the binding reaction was carried out in the absence of MgCl2, HutP failed to bind to hut mRNA, even in the presence of L-histidine. Thus, both Mg2+ ions and L-histidine are required to mediate the specific interactions between HutP and hut mRNA. Although Mg2+ ions are essential for the recognition by HutP of the specific sequence within the terminator region, their affinity for the HutP–L-histidine–RNA complex is unknown. Recently, a high-affinity binding site (Kd 489 mM) for Mg2+ ions was found within the ternary complex [22]. Furthermore, several different divalent metal ions can also mediate the HutP–RNA interaction, but these ions can not be replaced by monovalent cations.
Structure of the quaternary complex To gain insights into the importance of the interactions of HutP with L-histidine, RNA and Mg2+ ions, a quaternary complex structure was solved [20]. In this study, a shorter RNA (21-mer) possessing all of the conserved elements required for HutP recognition was used. HutP forms a hexameric structure, consisting of three individual dimers related by a crystallographic threefold axis, and recognizes the 21-mer RNA using both the top and bottom surfaces of the HutP protein, revealing a previously unreported triangular conformation (Figure 3a). The HutP hexamer recognizes two 21-mer RNA molecules, which is consistent with previous biochemical studies that detected a 1:2 (protein:RNA) complex ratio [18,20]. The crystallographic asymmetric unit of the quaternary complex contains one homodimer of HutP (molecules A and B), two L-histidines, two Mg2+ ions and two seven-nucleotide fragments of the 21-mer RNA, which are related by a non-crystallographic twofold axis (Figure 3b). Overall, the RNA structure adopts an extended A-RNA conformation, with C30 endo sugar conformations for all of the residues. The central bases, A4 and G5, are stacked on each other, and the remaining bases probably serve as spacers and do not interact with the protein molecule; their presence is apparently required to place the next UAG binding site on hexameric HutP. The 20 -OH groups of the A4 and U7 sugars form hydrogen bonds with the sidechains of Thr99 and Thr56, respectively. The importance of both contacts involving the 20 -OH groups is consistent with the results of the dexoyribonucleotide substitution and mutational analyses of the HutP protein [21]. This explains the ability of HutP to distinguish between RNA and DNA. The critical residues that www.sciencedirect.com
interact with the RNA — Glu55, Thr56, Thr99, Thr128 and Glu137 — were replaced with alanine; evaluation of their relative importance revealed that Thr99 and Glu137 were the most critical residues for RNA binding (Figure 3c). In contrast to the HutP–L-histidine analog complex, the L-histidines in the quaternary complex project downwards into the solvent; one imidazole nitrogen hydrogen bonds to a water molecule, while the other imidazole nitrogen hydrogen bonds to the sidechain oxygen of Tyr69 from the neighboring dimer within the HutP hexamer (Figure 3b,d). In the binary complex (HutP– L-histidine), the imidazole ring of L-histidine is buried in the hydrophobic pocket composed of residues from two adjacent molecules within the hexamer, in which Arg88 of molecule A and Glu81 of molecule B form a salt bridge (Figure 3e). The L-histidine analog, L-histidine bnaphthylamide, binds between the two molecules of HutP through four hydrogen bonds [two of them with b-strand residues of molecule A (Glu139 and Phe141) and two with a-helical residues of molecule B (Glu81 and Gly85)] and hydrophobic contacts with Phe141 of molecule A. Interestingly, in the quaternary complex, the amino and carboxyl groups of the L-histidine backbone participate in many interactions. The carboxyl group forms a typical salt bridge with the guanidyl group of Arg88. The amino and carboxyl groups coordinate the Mg2+ ion, which has a typical six-coordination sphere. Of the six coordinated ligands, three are the imidazole nitrogens of His138, His73 and His77. The latter two histidines belong to a neighboring dimer. The sixth ligand is a water molecule, which is anchored by a hydrogen bond to the sidechain of Glu81. These interactions were confirmed by mutational analyses and suggest the importance of the participation of Mg2+ ions in the interaction between HutP and L-histidine, along with residues from the specific binding site within the dimer and the dimer– dimer interface of HutP. The quaternary complex structure clearly established the importance of the residues involved in the various interactions: His73, His77 and His138 with the Mg2+ ion (Figure 3d); Tyr69, Glu81, Arg88 and Arg98 with the L-histidine (Figure 3d); and Glu55, Thr56, Thr99, Thr128 and Glu137 with the RNA (Figure 3c) [18,20]. The new HutP–L-histidine–Mg2+, HutP–L-histidine–Mn2+ and HutP–L-histidine–Ba2+ crystal structures revealed that the Mg2+-binding site can also accommodate different divalent cations, with ionic radii ranging from 0.72 to 1.35 A˚ [21].
Comparison of two complex structures to identify the structural rearrangement The overall structure of the quaternary complex of HutP resembles that of the HutP-L–histidine analog complex (Figure 4a); however, some significant structural changes were revealed, especially in the L-histidine-binding site (Figure 4b) and in loop regions L3 (Figure 4c) and L5 Current Opinion in Structural Biology 2006, 16:18–26
22 Protein–nucleic acid interactions
Figure 3
Overall structure of the HutP quaternary complex and the L-histidine-binding site. L-Histidine and RNA are shown as ball-and-stick models. Mg2+ ions and water molecules are represented by yellow and red spheres, respectively. (a) Surface potential representation of the quaternary complex and bound RNA. Basic regions are shown in blue and acidic regions in red. An alternative view of the hexamer is shown rotated by 908 along the y axis. (b) HutP dimer viewed along the pseudo-twofold axis. (c) Close-up view of the hydrogen-bonding interactions with RNA, shown by broken lines. The interactions of HutP with the bases and ribose moieties of A4 and G5 are illustrated. (d) Close-up view of the L-histidine- and Mg2+-binding sites of the quaternary complex. The hydrogen bonds are indicated by yellow dashed lines. (e) Close-up view of the L-histidine analog (HBN)-binding site of HutP; hydrogen bonds and salt bridges are indicated by yellow and red dashed lines, respectively.
(Figure 4d), upon HutP binding to L-histidine, divalent metal ions and RNA. On the basis of these two structures, it was difficult to pinpoint the specific ligand that causes the structural rearrangement of HutP. To identify both the ligand responsible for the structural rearrangement Current Opinion in Structural Biology 2006, 16:18–26
and the intermediate structures assumed by HutP as it attains the structure of the active anti-termination complex, two additional crystal structures of HutP were solved: apo-HutP in the presence of EDTA (Figure 2c), and in the presence of L-histidine and MgCl2 www.sciencedirect.com
HutP-mediated transcription anti-termination Kumar, Kumarevel and Mizuno 23
Figure 4
Stereo views of the conformational changes observed in the quaternary complex. The L-histidine analog, L-histidine and RNA are shown as ball-and-stick models, and the Mg2+ ions are represented by yellow spheres. Dotted black lines indicate hydrogen bonds. (a) Superposition of two crystal structures (HutP–L-histidine analog complex in purple; quaternary complex in green), showing the conformational changes of the L-histidine-binding site and the loop regions. (b) Comparison of the quaternary complex (green) and the HutP–L-histidine analog complex (purple), showing the structural differences around the L-histidine- and Mg2+-binding sites. Helix a4, at the bottom left, is derived from the neighboring dimer within the hexamer. (c) Conformational changes observed within loop L3, colored as in (b). (d) Conformational changes observed within loop L5, colored as in (b).
(Figure 2d). Comparing these two crystal structures of HutP revealed the details of the structural rearrangement, particularly in the L-histidine-binding site and loop regions L3 and L5 [20].
Structural rearrangement The structure of uncomplexed HutP (Figure 2c) is similar to the HutP–L-histidine (analog) complex structures, in terms of the existence of a hydrophobic pocket even in the absence of L-histidine. The HutP–L-histidine–Mg2+ structure (Figure 2d) closely resembles that of the quaternary complex (HutP–L-histidine– Mg2+–RNA), even despite the absence of RNA. This suggests that the RNA is not responsible for the observed structural rearrangewww.sciencedirect.com
ment, but that the metal ions and L-histidine ligand cause these changes before the target RNA is recognized. In these complexes, the metal ion and L-histidine mutually interact and facilitate the required overall structural rearrangement, because if even one of the two components (metal ion or L-histidine) is absent, HutP fails to bind to the RNA to form an anti-termination complex. However, L-histidine recognition must be the first step in this process, because until the imidazole ring of the L-histidine ligand is present in the hydrophobic pocket, HutP is not ready for the Mg2+-ion-mediated structural rearrangement. It seems that once the Mg2+ ion is recognized by the HutP–L-histidine complex, the Mg2+ ion might pull the L-histidine out of the hydrophobic pocket and bind to Current Opinion in Structural Biology 2006, 16:18–26
24 Protein–nucleic acid interactions
it tightly. In this way, the Mg2+-bound L-histidine could move along the dimer interface to a location 12 A˚ from the pocket. This L-histidine movement is apparently linked to the movement of Arg88 in the opposite direction. This leads to the disruption of the hydrophobic pocket, and the formation of a new salt bridge between Arg88 and
L-histidine
(Figure 4b). L-Histidine is also hydrogen bonded to Arg98 (Figure 4b), resulting in the drastic rearrangement of Arg98 (Ca position shifted by 5.4 A˚). This rearrangement is accompanied by a dramatic change in the Ca position of the next residue, Thr99 (5.0 A˚). Consequently, the Thr99 sidechain forms two hydrogen
Figure 5
Proposed schematic models of the activation of HutP and the existence of two potential HutP-binding sites. (a) Proposed steps undertaken by HutP before RNA binding. (b) Proposed model of the existence of two potential binding sites (highlighted by pink boxes) within the terminator region. The GC-rich region is highlighted by a black box. Current Opinion in Structural Biology 2006, 16:18–26
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HutP-mediated transcription anti-termination Kumar, Kumarevel and Mizuno 25
bonds, with the N3 and 20 -OH of the A4 base of the RNA (Figures 3c and 4c). On the other end, the reorientation of the His138 imidazole ring for coordination with the Mg2+ ion causes a large conformational change in the local backbone chain, particularly the torsion angles around His138 and the next residue, Glu137. As a result, the backbone oxygen of Glu137 bonds with the hydroxyl group of Tyr112. This changes the orientation of the sidechain of Glu137, allowing it to interact specifically with the G5 base through two hydrogen bonds (Figures 3c and 4d). These changes in HutP, initiated by the Mg2+ ion, might be transmitted along the backbone chain to Thr128 through the L5 loop. The sidechain of Thr128 thus moves to a suitable position to interact specifically with the A4 base through two hydrogen bonds (Figures 3c and 4d). Also, the backbone oxygen atoms of Ala131 and Pro132, located within the L5 loop, are involved in a hydrogen bond with the G5 base (Figure 3c) [20].
Mechanism of activation On the basis of analyses of the various structural and biochemical studies of HutP, we proposed rearranged structures of HutP for each stage of anti-termination. Initially, HutP exists as a hexamer, with a hydrophobic pocket in the center of each HutP dimer (Figure 5a, step A). In the next step (Figure 5a, step B), the incoming ligand is verified in the pocket, such that an imidazolecontaining residue is selected from others. L-Histidine interacts extensively with HutP residues within the pocket. Once the L-histidine interactions have been completed, HutP undergoes a significant structural rearrangement in the presence of Mg2+ ions or other divalent metal ions. Following this (Figure 5a, step C), the Lhistidine moves away from the hydrophobic pocket, and establishes a new interaction site with HutP and Mg2+ ions. This causes the L3 and L5 loops to specifically move and reorient residues Arg88, Thr99, Thr128, Glu137 and His138 within these loops. The reorientation of these residues is referred to as the activation process of HutP. Once HutP undergoes these conformational changes caused by L-histidine and Mg2+ ions, then it is ready to recognize the specific chemical groups of the bases within the hut mRNA, without undergoing any further structural rearrangement (Figure 5a, step D) [20].
Destabilization of the terminator structure The structural and biochemical studies presented here have explained how HutP undergoes a conformational rearrangement before binding the hut mRNA, but have not revealed how the stable terminator structure is subsequently altered and destabilized. In addition, what is the relevance of providing two surfaces (top and bottom) for RNA binding to HutP? To address these questions, the entire intervening region between the hutP gene stop codon and the initiation codon of the first structural gene, hutH, was reexamined, revealing the existence of two potential binding sites (site I, +498 to +514; site II, +535 to www.sciencedirect.com
+549). Each binding site contains three NAG motifs. One surface of hexameric HutP may occupy site I and the other surface site II. A 20-nucleotide spacer region is present between the two binding sites, which is sufficient to place site II on the other surface of hexameric HutP [20]. It is also interesting to observe that the HutPbinding sites are located upstream and downstream of the GC-rich region of the terminator (Figure 5b). The GCrich region can potentially form highly stable structures within the terminator regions; HutP binding either side of this region can potentially cause destabilization of this region. Although there is no direct evidence yet that the two binding sites are occupied by a hexameric HutP within the terminator, this has been proposed based on the existence of two potential binding sites within the hut mRNA and also the presence of two RNA-binding surfaces within HutP. The binding of hexameric HutP to these two sites would result in the complete destabilization of the terminator structure, thus allowing RNA polymerase to pass through the terminator to synthesize the full-length transcript.
Conclusions HutP, which mediates transcription anti-termination, uses a unique approach to regulate the expression of histidine utilization genes of the hut operon. Recent structural and biochemical studies have clearly explained how the HutP– RNA interactions are critically regulated by L-histidine and divalent metal ions, through structural rearrangements. Furthermore, by analyzing and comparing apoHutP, HutP–L-histidine analog, HutP–L-histidine–Mg2+ and HutP–L-histidine–Mg2+–RNA structures, we could identify the intermediate structures of HutP before the formation of activated HutP, which recognizes the hut mRNA. Nevertheless, further structural and biochemical studies are required to understand how the terminator structure is destabilized upon binding to activated HutP.
Acknowledgements This work was supported by funds from the National Institute of Industrial Science and Technology (AIST) to PKRK. TSK was supported by an AIST fellowship. We thank our colleague D Balasundaresan for help in preparing the figures.
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LicT transcriptional anti-terminator. EMBO J 2001, 20:3789-3799. 16. Yang Y, Declerck N, Manivel X, Aymerich S, Kochayan M: Solution structure of the LicT-RNA anti-termination complex: CAT clamping RAT. EMBO J 2002, 21:1987-1997. 17. Kumarevel TS, Fujimoto Z, Padmanabhan B, Oda M, Nishikawa S, Mizuno H, Kumar PKR: Crystallization and preliminary X-ray diffraction studies of HutP protein: an RNA-binding protein that regulates the transcription of hut operon in Bacillus subtilis. J Struct Biol 2002, 138:237-240. 18. Kumarevel T, Fujimoto Z, Karthe P, Oda M, Mizuno H, Kumar PKR: Crystal structure of activated HutP: an RNA binding protein that regulates transcription of the hut operon in Bacillus subtilis. Structure 2004, 12:1269-1280. The first reported crystal structure of HutP complexed with an l-histidine analog. The important chemical groups of l-histidine that enable HutP recognition of the cognate mRNA were identified. A minimal RNA motif containing three UAG motifs was suggested to be the core region for HutP binding. 19. Kumarevel TS, Mizuno H, Kumar PK: Allosteric activation of HutP protein, that regulates transcription of hut operon in Bacillus subtilis, mediated by various analogs of histidine. Nucleic Acids Res Suppl 2003, 3:199-200. 20. Kumarevel T, Mizuno H, Kumar PKR: Structural basis of HutP mediated anti-termination and roles of the Mg2+ ion and L-histidine ligand. Nature 2005, 434:183-191. This report revealed the quaternary complex structure of HutP and showed that the RNA adopts a novel triangular fold. The structure explains how the HutP–RNA interactions are regulated critically by Lhistidine and Mg2+ ions through structural rearrangement. In addition, two crystal structures (HutP and HutP–L-histidine–Mg2+) were solved and compared with the quaternary and previously reported [14] structures. These studies not only identified the intermediate structures of HutP, but also revealed the importance of the Mg2+ ion in the complexes. 21. Kumarevel TS, Gopinath SCB, Nishikawa S, Mizuno H, Kumar PKR: Identification of important chemical groups of the hut mRNA for HutP interactions that regulate the hut operon in Bacillus subtilis. Nucleic Acids Res 2004, 32:3904-3912. The important chemical groups of the RNA within the UAG motif were identified and the 1:2 molar ratio (protein:RNA) of HutP–RNA binding was determined. Furthermore, Glu137 of HutP was proposed to be very important for the HutP–hut mRNA interaction. 22. Kumarevel T, Mizuno H, Kumar PKR: Characterization of the metal ion binding site in the anti-terminator protein, HutP, of Bacillus subtilis. Nucleic Acids Res 2005, 33:5494-5502. This study showed that several different divalent metal ions can mediate the HutP protein–RNA interaction, but monovalent cations cannot. An efficient divalent metal ion binding pocket was identified.
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Current Opinion in Structural Biology 2006, 16:18–26 This review comes from a themed issue on Protein–nucleic acid interactions Edited by Gregory D Van Duyne and Wei Yang Available online 19th January 2006 0959-440X/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2006.01.005
Introduction Anti-terminator proteins control gene expression by recognizing control signals within cognate transcripts and preventing transcription termination. Examples of such regulatory proteins in Escherichia coli and Bacillus subtilis include BglG, SacT, SacY, TRAP and PyrR, which are known to regulate the bgl, sac, trp and pyr operons, respectively [1–5]. Many of these proteins exert their function as anti-terminators or attenuators upon binding to target sequences within nascent mRNAs. Another such protein is HutP, an anti-terminator protein that regulates the histidine utilization (hut) operon in B. subtilis [6,7]. The hut operon consists of five histidine utilization genes, hutH, hutU, hutI, hutG and hutM, which encode histidase, Current Opinion in Structural Biology 2006, 16:18–26
urocanase, imidazolone propionate amino hydrolase, formiminoglutamate hydrolase and histidine permease, respectively, and a positive regulatory gene, hutP (Figure 1a) [8–11]. The hutP gene is located just downstream of the hut promoter, whereas the histidine utilization genes are located far downstream of the promoter [10]. Expression of the operon is regulated by catabolite repression and by L-histidine-induced transcription antitermination. The mRNA transcript nucleotide sequence located between the hutP coding region and the coding region for the five histidine utilization genes is predicted to form a stem-loop structure that functions as a terminator (Figure 1b). In addition, a cis-acting regulatory sequence required for histidine-mediated induction of hut structural gene expression has been identified. It forms an anti-terminator structure and is located just upstream of the hut histidine utilization genes, partially overlapping the downstream terminator [7,12]. HutP is a 16.2 kDa protein consisting of 148 amino acid residues. HutP also exists in three other Bacillus species, including B. anthracis, B. cereus and B. halodurans, with 60% sequence identity. Sequence comparison of the Bacillus HutP proteins revealed that the C-terminal amino acid residues are more conserved than the Nterminal residues. Interestingly, the HutP protein binds to the terminator region within the hut mRNA and enhances hut structural gene expression only when activated by L-histidine. Several lines of evidence have indicated that HutP regulates the expression of the downstream genes of the hut operon by an anti-termination mechanism [6,7,12]. It is important to determine the conformational changes within HutP that allow it to recognize cognate sequences within the hut mRNA. Although other attenuator or anti-terminator proteins, such as TRAP [13,14] and LicT [15,16], form binary and ternary complexes, the conformational changes that the proteins undergo within these structures before binding to their cognate RNAs have remained obscure. Recently, the conformational changes that occur in HutP before cognate RNA binding have been described. Furthermore, these studies proposed how the terminator of the hut operon is destabilized by the activated HutP, facilitating the synthesis of the full-length transcript by RNA polymerase. The conformational changes induced by L-histidine and Mg2+ are of great interest, and are described in this review.
Structure of the HutP–L-histidine analog complex The first reported crystal structure of HutP was the HutP–L-histidine b-naphthylamide (L-histidine analog) www.sciencedirect.com
HutP-mediated transcription anti-termination Kumar, Kumarevel and Mizuno 19
Figure 1
The hut operon of B. subtilis. (a) Schematic representation of the hut operon, showing the arrangement of the hutP gene, terminator/anti-terminator region and histidine utilization genes. (b) The proposed hut mRNA terminator structure. Within the HutP-recognition motif, the UAG triplets are shown in pink. The stop codon of the hutP gene and the start codon of the hutH gene are shown in green.
complex structure, referred to in this review as the HutP– L-histidine analog complex [17,18 ]. In this complex, the HutP protein adopts an a/b structure, consisting of four a helices and four b strands. These are arranged in the order a-a-b-a-a-b-b-b in the primary structure; the four antiparallel b strands form a b sheet in the order b1b2-b3-b4, with two a helices each at the front and back of the b sheet. Three HutP dimers (molecules A and B related by non-crystallographic twofold symmetry) are related by threefold symmetry to form a hexamer, which seems to be the functional form [18] (Figure 2a,b). The apparent molecular weight of HutP, as determined by native gel studies, suggested that it exists as a hexamer [12]. The hexameric structure is stabilized by hydrophobic contacts between the dimers, especially residues 68– 80 of molecule A, which form many hydrophobic contacts with the neighboring dimer. Similarly, residues 90–98 and 131–136 of molecule B contact residues 73–84 and 46–53, respectively, of neighboring molecule B. The overall structure of HutP is unrelated to any structures currently available in the PDB, including known RNA-binding proteins, and thus it represents a novel RNA-binding motif. www.sciencedirect.com
HutP binds to its cognate terminator RNA only in the presence of L-histidine; it requires about 10 mM of Lhistidine for complete activation [12,18]. Therefore, it is believed that L-histidine allosterically controls HutP–hut mRNA interactions by modifying the conformation of HutP. The chemical groups of L-histidine responsible for activating the HutP protein have been determined by binding studies with various analogs of L-histidine [19]. Among these, the best analogs that supported HutP activation and binding to the hut mRNA were found to be L-histidine, L-histidine benzyl ester and L-histidine bnaphthylamide. These studies led to the conclusion that the imidazole group and the backbone of L-histidine are essential for the HutP activation process. To determine the details of the interaction with HutP, crystal structures of the HutP–L-histidine, HutP–L-histidine benzyl ester and HutP–L-histidine b-naphthylamide complexes have been solved [18]. These studies revealed that a hydrophobic pocket stabilizes the dimer interface through the formation of a salt bridge between the HutP monomers, involving Glu81 of molecule B and Arg88 of molecule A. L-Histidine binds within this hydrophobic pocket. The importance of these residues for mediating the interaction Current Opinion in Structural Biology 2006, 16:18–26
20 Protein–nucleic acid interactions
Figure 2
Structures of HutP complexes. (a) HutP hexamer viewed along the threefold axis, with the L-histidine analog (L-histidine b-naphthylamide, HBN) shown in pink as a ball-and-stick model. (b) HutP dimer viewed along the non-crystallographic twofold axis. HutP is shown as a ribbon diagram, with labeled a helices, b strands and loops (molecule A blue, molecule B green). (c) Dimer of apo-HutP. (d) Dimer of the ternary complex (HutP–L-histidine–Mg2+ ion). The bound L-histidine and Mg2+ ion are shown as a ball-and-stick model and a yellow sphere, respectively.
with a specific sequence within the hut mRNA, through their interaction with the L-histidine analog, was confirmed by site-specific mutational studies [18]. Recently, we have also solved two additional crystal structures, apo-HutP (Figure 2c) and the HutP–L-histidine–Mg2+ ternary complex (Figure 2d), to further understand the conformational changes that HutP is believed to undergo before binding to its cognate target site within hut mRNA [20]; a description of these structures will follow.
The minimal RNA motif and important chemical groups Initially, a minimal RNA region sufficient for efficient HutP binding was mapped to the region encompassing nucleotides +459 to +537 (79-mer RNA) of the hut mRNA [12]. As HutP exists as a hexamer, as mentioned above, a search for repeated sequences within the mapped region was made. Interestingly, three UAG repeats separated by Current Opinion in Structural Biology 2006, 16:18–26
four-nucleotide spacers were found. A short oligomer (spanning +496 to +515) was prepared, which bound as efficiently as the 79-mer RNA. Next, to address the importance of the spacer region to HutP binding, the spacer nucleotides were substituted by U bases, which revealed that the chemical groups of the spacer bases are not important, but that the presence of four nucleotides in the spacer is optimal for positioning the next binding site in the context of the hexamer [18]. To determine the RNA chemical groups that are essential for the interaction with HutP, an in vitro selection strategy and analyses of site-specific base substitutions were carried out [21]. These studies suggested that each HutP molecule recognizes one UAG motif; the first base (U) can be substituted by other bases, whereas the second and third bases (A and G) are essential for the interaction (NAG motif). Analyses of the chemical groups of the A and G bases within the UAG motif, using modified base analogs, suggested the importance of the exocyclic NH2 groups. In addition, in www.sciencedirect.com
HutP-mediated transcription anti-termination Kumar, Kumarevel and Mizuno 21
this motif, only the 20 -OH group of the A base is important for HutP recognition [18].
Divalent metal ion requirement All of the above studies showed that L-histidine is required for HutP to recognize the cognate target sequence within the hut mRNA. However, these studies were performed in a binding buffer containing 10 mM MgCl2. When the binding reaction was carried out in the absence of MgCl2, HutP failed to bind to hut mRNA, even in the presence of L-histidine. Thus, both Mg2+ ions and L-histidine are required to mediate the specific interactions between HutP and hut mRNA. Although Mg2+ ions are essential for the recognition by HutP of the specific sequence within the terminator region, their affinity for the HutP–L-histidine–RNA complex is unknown. Recently, a high-affinity binding site (Kd 489 mM) for Mg2+ ions was found within the ternary complex [22]. Furthermore, several different divalent metal ions can also mediate the HutP–RNA interaction, but these ions can not be replaced by monovalent cations.
Structure of the quaternary complex To gain insights into the importance of the interactions of HutP with L-histidine, RNA and Mg2+ ions, a quaternary complex structure was solved [20]. In this study, a shorter RNA (21-mer) possessing all of the conserved elements required for HutP recognition was used. HutP forms a hexameric structure, consisting of three individual dimers related by a crystallographic threefold axis, and recognizes the 21-mer RNA using both the top and bottom surfaces of the HutP protein, revealing a previously unreported triangular conformation (Figure 3a). The HutP hexamer recognizes two 21-mer RNA molecules, which is consistent with previous biochemical studies that detected a 1:2 (protein:RNA) complex ratio [18,20]. The crystallographic asymmetric unit of the quaternary complex contains one homodimer of HutP (molecules A and B), two L-histidines, two Mg2+ ions and two seven-nucleotide fragments of the 21-mer RNA, which are related by a non-crystallographic twofold axis (Figure 3b). Overall, the RNA structure adopts an extended A-RNA conformation, with C30 endo sugar conformations for all of the residues. The central bases, A4 and G5, are stacked on each other, and the remaining bases probably serve as spacers and do not interact with the protein molecule; their presence is apparently required to place the next UAG binding site on hexameric HutP. The 20 -OH groups of the A4 and U7 sugars form hydrogen bonds with the sidechains of Thr99 and Thr56, respectively. The importance of both contacts involving the 20 -OH groups is consistent with the results of the dexoyribonucleotide substitution and mutational analyses of the HutP protein [21]. This explains the ability of HutP to distinguish between RNA and DNA. The critical residues that www.sciencedirect.com
interact with the RNA — Glu55, Thr56, Thr99, Thr128 and Glu137 — were replaced with alanine; evaluation of their relative importance revealed that Thr99 and Glu137 were the most critical residues for RNA binding (Figure 3c). In contrast to the HutP–L-histidine analog complex, the L-histidines in the quaternary complex project downwards into the solvent; one imidazole nitrogen hydrogen bonds to a water molecule, while the other imidazole nitrogen hydrogen bonds to the sidechain oxygen of Tyr69 from the neighboring dimer within the HutP hexamer (Figure 3b,d). In the binary complex (HutP– L-histidine), the imidazole ring of L-histidine is buried in the hydrophobic pocket composed of residues from two adjacent molecules within the hexamer, in which Arg88 of molecule A and Glu81 of molecule B form a salt bridge (Figure 3e). The L-histidine analog, L-histidine bnaphthylamide, binds between the two molecules of HutP through four hydrogen bonds [two of them with b-strand residues of molecule A (Glu139 and Phe141) and two with a-helical residues of molecule B (Glu81 and Gly85)] and hydrophobic contacts with Phe141 of molecule A. Interestingly, in the quaternary complex, the amino and carboxyl groups of the L-histidine backbone participate in many interactions. The carboxyl group forms a typical salt bridge with the guanidyl group of Arg88. The amino and carboxyl groups coordinate the Mg2+ ion, which has a typical six-coordination sphere. Of the six coordinated ligands, three are the imidazole nitrogens of His138, His73 and His77. The latter two histidines belong to a neighboring dimer. The sixth ligand is a water molecule, which is anchored by a hydrogen bond to the sidechain of Glu81. These interactions were confirmed by mutational analyses and suggest the importance of the participation of Mg2+ ions in the interaction between HutP and L-histidine, along with residues from the specific binding site within the dimer and the dimer– dimer interface of HutP. The quaternary complex structure clearly established the importance of the residues involved in the various interactions: His73, His77 and His138 with the Mg2+ ion (Figure 3d); Tyr69, Glu81, Arg88 and Arg98 with the L-histidine (Figure 3d); and Glu55, Thr56, Thr99, Thr128 and Glu137 with the RNA (Figure 3c) [18,20]. The new HutP–L-histidine–Mg2+, HutP–L-histidine–Mn2+ and HutP–L-histidine–Ba2+ crystal structures revealed that the Mg2+-binding site can also accommodate different divalent cations, with ionic radii ranging from 0.72 to 1.35 A˚ [21].
Comparison of two complex structures to identify the structural rearrangement The overall structure of the quaternary complex of HutP resembles that of the HutP-L–histidine analog complex (Figure 4a); however, some significant structural changes were revealed, especially in the L-histidine-binding site (Figure 4b) and in loop regions L3 (Figure 4c) and L5 Current Opinion in Structural Biology 2006, 16:18–26
22 Protein–nucleic acid interactions
Figure 3
Overall structure of the HutP quaternary complex and the L-histidine-binding site. L-Histidine and RNA are shown as ball-and-stick models. Mg2+ ions and water molecules are represented by yellow and red spheres, respectively. (a) Surface potential representation of the quaternary complex and bound RNA. Basic regions are shown in blue and acidic regions in red. An alternative view of the hexamer is shown rotated by 908 along the y axis. (b) HutP dimer viewed along the pseudo-twofold axis. (c) Close-up view of the hydrogen-bonding interactions with RNA, shown by broken lines. The interactions of HutP with the bases and ribose moieties of A4 and G5 are illustrated. (d) Close-up view of the L-histidine- and Mg2+-binding sites of the quaternary complex. The hydrogen bonds are indicated by yellow dashed lines. (e) Close-up view of the L-histidine analog (HBN)-binding site of HutP; hydrogen bonds and salt bridges are indicated by yellow and red dashed lines, respectively.
(Figure 4d), upon HutP binding to L-histidine, divalent metal ions and RNA. On the basis of these two structures, it was difficult to pinpoint the specific ligand that causes the structural rearrangement of HutP. To identify both the ligand responsible for the structural rearrangement Current Opinion in Structural Biology 2006, 16:18–26
and the intermediate structures assumed by HutP as it attains the structure of the active anti-termination complex, two additional crystal structures of HutP were solved: apo-HutP in the presence of EDTA (Figure 2c), and in the presence of L-histidine and MgCl2 www.sciencedirect.com
HutP-mediated transcription anti-termination Kumar, Kumarevel and Mizuno 23
Figure 4
Stereo views of the conformational changes observed in the quaternary complex. The L-histidine analog, L-histidine and RNA are shown as ball-and-stick models, and the Mg2+ ions are represented by yellow spheres. Dotted black lines indicate hydrogen bonds. (a) Superposition of two crystal structures (HutP–L-histidine analog complex in purple; quaternary complex in green), showing the conformational changes of the L-histidine-binding site and the loop regions. (b) Comparison of the quaternary complex (green) and the HutP–L-histidine analog complex (purple), showing the structural differences around the L-histidine- and Mg2+-binding sites. Helix a4, at the bottom left, is derived from the neighboring dimer within the hexamer. (c) Conformational changes observed within loop L3, colored as in (b). (d) Conformational changes observed within loop L5, colored as in (b).
(Figure 2d). Comparing these two crystal structures of HutP revealed the details of the structural rearrangement, particularly in the L-histidine-binding site and loop regions L3 and L5 [20].
Structural rearrangement The structure of uncomplexed HutP (Figure 2c) is similar to the HutP–L-histidine (analog) complex structures, in terms of the existence of a hydrophobic pocket even in the absence of L-histidine. The HutP–L-histidine–Mg2+ structure (Figure 2d) closely resembles that of the quaternary complex (HutP–L-histidine– Mg2+–RNA), even despite the absence of RNA. This suggests that the RNA is not responsible for the observed structural rearrangewww.sciencedirect.com
ment, but that the metal ions and L-histidine ligand cause these changes before the target RNA is recognized. In these complexes, the metal ion and L-histidine mutually interact and facilitate the required overall structural rearrangement, because if even one of the two components (metal ion or L-histidine) is absent, HutP fails to bind to the RNA to form an anti-termination complex. However, L-histidine recognition must be the first step in this process, because until the imidazole ring of the L-histidine ligand is present in the hydrophobic pocket, HutP is not ready for the Mg2+-ion-mediated structural rearrangement. It seems that once the Mg2+ ion is recognized by the HutP–L-histidine complex, the Mg2+ ion might pull the L-histidine out of the hydrophobic pocket and bind to Current Opinion in Structural Biology 2006, 16:18–26
24 Protein–nucleic acid interactions
it tightly. In this way, the Mg2+-bound L-histidine could move along the dimer interface to a location 12 A˚ from the pocket. This L-histidine movement is apparently linked to the movement of Arg88 in the opposite direction. This leads to the disruption of the hydrophobic pocket, and the formation of a new salt bridge between Arg88 and
L-histidine
(Figure 4b). L-Histidine is also hydrogen bonded to Arg98 (Figure 4b), resulting in the drastic rearrangement of Arg98 (Ca position shifted by 5.4 A˚). This rearrangement is accompanied by a dramatic change in the Ca position of the next residue, Thr99 (5.0 A˚). Consequently, the Thr99 sidechain forms two hydrogen
Figure 5
Proposed schematic models of the activation of HutP and the existence of two potential HutP-binding sites. (a) Proposed steps undertaken by HutP before RNA binding. (b) Proposed model of the existence of two potential binding sites (highlighted by pink boxes) within the terminator region. The GC-rich region is highlighted by a black box. Current Opinion in Structural Biology 2006, 16:18–26
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HutP-mediated transcription anti-termination Kumar, Kumarevel and Mizuno 25
bonds, with the N3 and 20 -OH of the A4 base of the RNA (Figures 3c and 4c). On the other end, the reorientation of the His138 imidazole ring for coordination with the Mg2+ ion causes a large conformational change in the local backbone chain, particularly the torsion angles around His138 and the next residue, Glu137. As a result, the backbone oxygen of Glu137 bonds with the hydroxyl group of Tyr112. This changes the orientation of the sidechain of Glu137, allowing it to interact specifically with the G5 base through two hydrogen bonds (Figures 3c and 4d). These changes in HutP, initiated by the Mg2+ ion, might be transmitted along the backbone chain to Thr128 through the L5 loop. The sidechain of Thr128 thus moves to a suitable position to interact specifically with the A4 base through two hydrogen bonds (Figures 3c and 4d). Also, the backbone oxygen atoms of Ala131 and Pro132, located within the L5 loop, are involved in a hydrogen bond with the G5 base (Figure 3c) [20].
Mechanism of activation On the basis of analyses of the various structural and biochemical studies of HutP, we proposed rearranged structures of HutP for each stage of anti-termination. Initially, HutP exists as a hexamer, with a hydrophobic pocket in the center of each HutP dimer (Figure 5a, step A). In the next step (Figure 5a, step B), the incoming ligand is verified in the pocket, such that an imidazolecontaining residue is selected from others. L-Histidine interacts extensively with HutP residues within the pocket. Once the L-histidine interactions have been completed, HutP undergoes a significant structural rearrangement in the presence of Mg2+ ions or other divalent metal ions. Following this (Figure 5a, step C), the Lhistidine moves away from the hydrophobic pocket, and establishes a new interaction site with HutP and Mg2+ ions. This causes the L3 and L5 loops to specifically move and reorient residues Arg88, Thr99, Thr128, Glu137 and His138 within these loops. The reorientation of these residues is referred to as the activation process of HutP. Once HutP undergoes these conformational changes caused by L-histidine and Mg2+ ions, then it is ready to recognize the specific chemical groups of the bases within the hut mRNA, without undergoing any further structural rearrangement (Figure 5a, step D) [20].
Destabilization of the terminator structure The structural and biochemical studies presented here have explained how HutP undergoes a conformational rearrangement before binding the hut mRNA, but have not revealed how the stable terminator structure is subsequently altered and destabilized. In addition, what is the relevance of providing two surfaces (top and bottom) for RNA binding to HutP? To address these questions, the entire intervening region between the hutP gene stop codon and the initiation codon of the first structural gene, hutH, was reexamined, revealing the existence of two potential binding sites (site I, +498 to +514; site II, +535 to www.sciencedirect.com
+549). Each binding site contains three NAG motifs. One surface of hexameric HutP may occupy site I and the other surface site II. A 20-nucleotide spacer region is present between the two binding sites, which is sufficient to place site II on the other surface of hexameric HutP [20]. It is also interesting to observe that the HutPbinding sites are located upstream and downstream of the GC-rich region of the terminator (Figure 5b). The GCrich region can potentially form highly stable structures within the terminator regions; HutP binding either side of this region can potentially cause destabilization of this region. Although there is no direct evidence yet that the two binding sites are occupied by a hexameric HutP within the terminator, this has been proposed based on the existence of two potential binding sites within the hut mRNA and also the presence of two RNA-binding surfaces within HutP. The binding of hexameric HutP to these two sites would result in the complete destabilization of the terminator structure, thus allowing RNA polymerase to pass through the terminator to synthesize the full-length transcript.
Conclusions HutP, which mediates transcription anti-termination, uses a unique approach to regulate the expression of histidine utilization genes of the hut operon. Recent structural and biochemical studies have clearly explained how the HutP– RNA interactions are critically regulated by L-histidine and divalent metal ions, through structural rearrangements. Furthermore, by analyzing and comparing apoHutP, HutP–L-histidine analog, HutP–L-histidine–Mg2+ and HutP–L-histidine–Mg2+–RNA structures, we could identify the intermediate structures of HutP before the formation of activated HutP, which recognizes the hut mRNA. Nevertheless, further structural and biochemical studies are required to understand how the terminator structure is destabilized upon binding to activated HutP.
Acknowledgements This work was supported by funds from the National Institute of Industrial Science and Technology (AIST) to PKRK. TSK was supported by an AIST fellowship. We thank our colleague D Balasundaresan for help in preparing the figures.
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LicT transcriptional anti-terminator. EMBO J 2001, 20:3789-3799. 16. Yang Y, Declerck N, Manivel X, Aymerich S, Kochayan M: Solution structure of the LicT-RNA anti-termination complex: CAT clamping RAT. EMBO J 2002, 21:1987-1997. 17. Kumarevel TS, Fujimoto Z, Padmanabhan B, Oda M, Nishikawa S, Mizuno H, Kumar PKR: Crystallization and preliminary X-ray diffraction studies of HutP protein: an RNA-binding protein that regulates the transcription of hut operon in Bacillus subtilis. J Struct Biol 2002, 138:237-240. 18. Kumarevel T, Fujimoto Z, Karthe P, Oda M, Mizuno H, Kumar PKR: Crystal structure of activated HutP: an RNA binding protein that regulates transcription of the hut operon in Bacillus subtilis. Structure 2004, 12:1269-1280. The first reported crystal structure of HutP complexed with an l-histidine analog. The important chemical groups of l-histidine that enable HutP recognition of the cognate mRNA were identified. A minimal RNA motif containing three UAG motifs was suggested to be the core region for HutP binding. 19. Kumarevel TS, Mizuno H, Kumar PK: Allosteric activation of HutP protein, that regulates transcription of hut operon in Bacillus subtilis, mediated by various analogs of histidine. Nucleic Acids Res Suppl 2003, 3:199-200. 20. Kumarevel T, Mizuno H, Kumar PKR: Structural basis of HutP mediated anti-termination and roles of the Mg2+ ion and L-histidine ligand. Nature 2005, 434:183-191. This report revealed the quaternary complex structure of HutP and showed that the RNA adopts a novel triangular fold. The structure explains how the HutP–RNA interactions are regulated critically by Lhistidine and Mg2+ ions through structural rearrangement. In addition, two crystal structures (HutP and HutP–L-histidine–Mg2+) were solved and compared with the quaternary and previously reported [14] structures. These studies not only identified the intermediate structures of HutP, but also revealed the importance of the Mg2+ ion in the complexes. 21. Kumarevel TS, Gopinath SCB, Nishikawa S, Mizuno H, Kumar PKR: Identification of important chemical groups of the hut mRNA for HutP interactions that regulate the hut operon in Bacillus subtilis. Nucleic Acids Res 2004, 32:3904-3912. The important chemical groups of the RNA within the UAG motif were identified and the 1:2 molar ratio (protein:RNA) of HutP–RNA binding was determined. Furthermore, Glu137 of HutP was proposed to be very important for the HutP–hut mRNA interaction. 22. Kumarevel T, Mizuno H, Kumar PKR: Characterization of the metal ion binding site in the anti-terminator protein, HutP, of Bacillus subtilis. Nucleic Acids Res 2005, 33:5494-5502. This study showed that several different divalent metal ions can mediate the HutP protein–RNA interaction, but monovalent cations cannot. An efficient divalent metal ion binding pocket was identified.
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