Structural modeling of RNase P RNA of the hyperthermophilic archaeon Pyrococcus horikoshii OT3

Biochemical and Biophysical Research Communications 414 (2011) 517–522

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Structural modeling of RNase P RNA of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 Christian Zwieb a, Yuji Nakao b, Takashi Nakashima b, Hisanori Takagi b, Shuichiro Goda c, Ebbe Sloth Andersen d, Yoshimitsu Kakuta b, Makoto Kimura b,⇑ a

Department of Biochemistry, University of Texas Health Science Center at San Antonio San Antonio, TX 78229-3900, USA Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Hakozaki 6-10-1, Fukuoka 812-8581, Japan Department of Applied Chemistry, Faculty of Engineering, Nagasaki University, Bunkyo-machi 1-14, Nagasaki 852-8521, Japan d Interdisciplinary Nanoscience Center, Department of Molecular Biology, Aarhus University, C.F. Møllers Allé 3, DK-8000 Aarhus, Denmark b c

a r t i c l e

i n f o

Article history: Received 15 September 2011 Available online 24 September 2011 Keywords: 3-D Reconstruction Comparative sequence analysis Pyrococcus horikoshii RNase P RNA structure

a b s t r a c t Ribonuclease P (RNase P) is a ubiquitous trans-acting ribozyme that processes the 50 leader sequence of precursor tRNA (pre-tRNA). The RNase P RNA (PhopRNA) of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 is central to the catalytic process and binds five proteins (PhoPop5, PhoRpp21, PhoRpp29, PhoRpp30, and PhoRpp38) which contribute to the enzymatic activity of the holoenzyme. Despite significant progress in determining the crystal structure of the proteins, the structure of PhopRNA remains elusive. Comparative analysis of the RNase P RNA sequences and existing crystallographic structural information of the bacterial RNase P RNAs were combined to generate a phylogenetically supported three-dimensional (3-D) model of the PhopRNA. The model structure shows an essentially flat disk with 16 tightly packed helices and a conserved face suitable for the binding of pre-tRNA. Moreover, the structure in solution was investigated by enzymatic probing and small-angle X-ray scattering (SAXS) analysis. The low resolution model derived from SAXS and the comparative 3-D model have similar overall shapes. The 3-D model provides a framework for a better understanding of structure–function relationships of this multifaceted primordial ribozyme. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction Ribonuclease P (RNase P) is an endoribonucleolytic trans-acting ribozyme required for the correct processing of tRNA precursors (pre-tRNA) into their 50 -phosphorylated mature forms [1]. In all domains of life, RNase P is composed of an RNA component and a variable number of RNase P proteins [2,3]. Since Altman and co-workers discovered that the Escherichia coli RNase P RNA (M1RNA) itself can hydrolyze pre-tRNA in vitro [4], biochemical and structural studies on RNase P have been focusing on the bacterial enzymes [5,6]. The three-dimensional RNase P RNA structures of Thermotoga maritima (bacterial Type A RNase P) and Bacillus stearothermophilus (bacterial Type B RNase P) have been solved [7,8], and structures of the bacterial protein subunit have been determined at high resolutions [9–11]. Very recently, the X-ray

Abbreviations: 3-D, three-dimensional; PhopRNA, P. horikoshii RNase P RNA; pretRNA, precursor tRNA; RNase P, ribonuclease P; SAXS, small-angle X-ray scattering. ⇑ Corresponding author. Fax: +81 92 642 2853. E-mail address: [email protected] (M. Kimura). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.09.098

structure of the T. maritima RNase P in complex with tRNA has been determined and a mechanism explaining the catalytic activity of the bacterial RNase P has been proposed [12]. In contrast to bacterial RNase P RNAs, archaeal and eukaryal RNase P RNAs alone have little enzymatic activity and function cooperatively with their cognate protein subunits in catalysis [13]. Therefore, for a full understanding of the molecular mechanisms of these molecules, it is a prerequisite to elucidate the three dimensional structure of the holoenzymes. We earlier found in reconstitution experiment that RNase P RNA (PhopRNA) of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 is enzymatically active only when bound to proteins PhoPop5, PhoRpp38, PhoRpp21, PhoRpp29, and PhoRpp30 [14,15]. As the crystal structures of the five P. horikoshii OT3 RNase P proteins have been determined [16] and references therein, there is a compelling need to determine the three-dimensional (3-D) structure of PhopRNA. Because crystals of the PhopRNA are unavailable, we combined sequence and structure comparisons to reconstruct the 3-D model of the PhopRNA. Moreover, we have measured the small-angle X-ray scattering (SAXS) profiles of PhopRNA to obtain the modeled structure by ab initio method using scattering curve.

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2. Materials and methods 2.1. Identification and aligning RNase P RNA sequences The initial set of RNase P RNA sequences was extracted from the Rfam site [17] using Perl scripts (available from the first author be request). The sequences were used as queries for a locally installed version of BLAST [18] to identify additional sequences. The four groupings of RNase P RNA sequences (Archaea, Bacteria Type, Bacteria Type B, and nuclear Eukaryota) were merged into one preliminary alignment that was based on sequence similarity using MUSCLE [19]. SARSE [20], a semiautomated procedure for improving the alignment by assigning compensatory base changes (CBCs) of the Watson–Crick type (G–C, A–U, and G–U) was used to generate a single pairing mask for all known RNase P RNAs using the previously define stringent rules [21]. In addition, Mfold [22] was used to identify a double-stranded region in PhopRNA. The alignment, as well as phylogenetically and alphabetically sorted organism lists and RNase P RNA secondary structures in the Connect format for every sequence in the database, are available at http://rnp.uthscsa.edu or http://www.bbs1.agr.kyushu-u.ac.jp/biosci-biotech/seibutu/rnp/RNasePDB/RNasePDB.html. 2.2. 3-D modeling of PhopRNA The 3-D model of the PhopRNA was reconstructed using version 1.2 of the program ERNA-3D [23], available at the internet address http://www.erna-3d.de/, on a SiliconGraphics Octane 2 workstation (IRIX 6.5). ERNA-3D’s inbuilt tools and functions were used to incorporate features extracted from the coordinates of the B. stearothermophilus (2a64.pdb) [7] and T. maritima (2a2e.pdb) [8] RNase P RNA crystal structures, as well as the Kt-7 K-turn of Haloarcula marismortui 23S rRNA (1ffk.pdb) [24,25]. For details see Supplementary Table 1. The coordinates of the 3-D model of PhopRNA are available at http://rnp.uthscsa.edu or http://bbs1.agr.kyushu-u.ac.jp/ biosci-biotech/seibutu/rnp/RNasePDB/RNasePDB.html. 2.3. Structural probing PhopRNA was transcribed in vitro, as described previously [14], and purified by ion-exchange column chromatography on a HiTrap DEAE-Sepharose FF column (GE Healthcare), as described by Easton et al. [26]. Enzymatic probing of the structure of PhopRNA was done using a mixture of RNase T1 and A, essentially as described by Stern et al. [27]. Five microgram of PhopRNA in reconstitution buffer (50 mM Tris–HCl, pH 7.5, 50 mM MgCl2, 800 mM CH3COONH4, 60 mM NH4Cl) was incubated at 50 °C for 30 min. Four microliter of RNase mixture (1.25 lg/ml RNase A and 25 lg/ml RNase T1 in TE buffer) was added to the appropriate tubes and incubated for 1 h on ice. The digestion was stopped by phenol extract, precipitated with ethanol and resuspended in 20 ll of water. The cleavage sites were identified by primer extension using 50 -[32P]-labeled four oligonucleotide primers P1, P2, P3, and P4, which were complementary to positions 296–316, 255–275, 204–224, and 91–111 along the PhopRNA chain, respectively. 2.4. Small-angle X-ray scattering (SAXS) The purified PhopRNA was dissolved in the reconstitution buffer and heated at 90 °C for 3 min, and then cooled to room temperature for 20 min. SAXS measurements were performed using the optics and detector system installed at beamline BL-10C of the 2.5 GeV-storage ring at the Photon Factory (Tsukuba Japan), as described by Higurashi et al. [28]. The radius of gyration Rg was

computed from the scattering patterns using the program GNOM [29], which provides the maximum particle dimensions Dmax and the distance distribution function P(r). Scattering curves of the coordinates were calculated using the program CRYSOL [30]. 2.5. Saxs ab initio reconstruction The low resolution shape of PhopRNA was reconstructed by the program DAMMIN [31] which represents particle shape as an ensemble of densely packed beads inside a spherical search volume with diameter Dmax. Starting from a random distribution of beads, simulated annealing was employed to find a compact configuration minimizing the discrepancy v between the experimental and calculated scattering curves [31]. 3. Results 3.1. Derivation of the PhopRNA secondary structures In order to establish the phylogenetically supported PhopRNA secondary structure, we used tools of the editor SARSE to merge the RNase P RNA sequences of the four Rfam groups: archaeal, bacterial Type A, bacterial Type B, and nuclear. The improved alignment contained a total of 844 RNase P RNA sequences, including 476 bacterial A-type RNAs, 162 bacterial B-type, 97 archaeal RNAs, and 109 nuclear RNAs. A single base pairing mask was generated to assign the helical regions as described in Section 2. The previously established procedures for the determination of compensatory Watson–Crick and G-U base changes identified 16 helices (P1–P16) in PhopRNA, which were numbered according to the existing RNase P RNA nomenclature [32] (Fig. 1A). The single-stranded loops between helices were designated the prefix J (e.g., the single-stranded loop, G233–A238, between helices P15 and P16 is referred to as J15/16 in Supplementary Table 1). The deduced base paired regions shown in Fig. 1A agreed well with previous assessments of the foldings of the archaeal type A RNase P RNAs [33,34]. Newly assigned were a K-turn motif between helices P12.1 and P12.2, and several bulged residues (Supplementary Fig. 1). The secondary structure model included the base pairs forming helix P11 (residues 100– 104 paired to 210–214) and helix P12.1 (residues 131–135 paired to 178–182). The latter helix was neither sufficiently supported nor unsupported by comparative sequence analysis but was predicted by Mfold [22]. 3.2. 3-D modeling ERNA-3D was used in order to create a three-dimensional representation of the PhopRNA from the sequence and base pairing information. The initial model depicted the helical sections in the A-form of the RNA. The conformations of the single-stranded regions were calculated by ERNA-3D’s inbuilt algorithm. To model the conserved core of the PhopRNA we copied the coordinates of the homologous regions of the bacterial RNase P RNAs (Supplementary Table 1), including the RNase P RNA crystal structures of T. martitima (2a2e.pdb) [8] and B. stearothermophilus (2a64.pdb) [7]. The K-turn between helices P12.1 and P12.2 was modeled according to the Kt-7 element in 23S rRNA [25]. The RNA backbone connections between the motif and the rest of the model were inspected and, if needed, adjustments were made to correct bond lengths and tetrahedral angles involving the phosphorous atom at the joint between the extracted motif and helical structures to arrive at the final reconstruction. Overall, the 3-D model of the PhopRNA conforms to an essentially flat disk of tightly packed helices with the approximate dimensions

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Fig. 1. 3-D model of PhopRNA. (A) Secondary structure of PhopRNA. Shown are the 50 and 30 ends, Watson–Crick base pairs by letter-connecting short lines, and G–U pairs by dots. Residues are numbered in increments of 10. Helices are numbered according to the existing RNase P RNA nomenclature [32]. Brackets connected by lines mark helices 4 and 6. The K-turn motif consensus [25] is shown within the box below the junction of helices P12.1 and P12.2. Helices are colored and single-stranded loops are dark gray. (B) Two views of the PhopRNA 3D model. (Top) View into the presumed tRNA binding surface. (Bottom) View from the top showing the arrangement of the tightly packed helices. The sugar–phosphate backbone is represented by a ribbon with the same color scheme as in (A). Figures were drawn with PyMol (http://pymol.sourceforge.net). (For interpretation of the references to colors in this figure legend, the reader is referred to the web version of this paper.)

110 Å  95 Å  20 Å, as presented in Fig. 1B. In the model structure, the co-axially stacked helices P8/P9 are located at the center of the molecule with the co-axial helices P12/P12.1 and P10/P11 in the S-domain on one side and the stacked helices P2/P3 and P1/P4/P5 in the C-domain on the other side. The modeled phylogenetically well-supported K-turn between helices P12.1 and P12.2 enforces compact folding of this more variable region and may bind to PhoRpp38. Surrounding helix P12.2, additional RNA segments are observed in the Sulfolobus acidocaldarius sequence (Supplementary Fig. 2). These elements could be placed towards the ‘‘backside’’ of the model without perturbing the catalytic face. 3.3. Enzymatic probing To evaluate the proposed structural model and gain information about the PhopRNA in solution, we carried out enzymatic probing using a mixture of RNases T1 and A. A representative autoradiogram from the enzymatic probing experiments is shown in Fig. 2A. Results are summarized in Fig. 2B and superimposed on a schematic diagram of the proposed PhopRNA secondary structure. Inspection of Fig. 2B shows that in general the proposed structure was well supported but some contradictions were observed. G145 was sensitive to the digestion, supporting the presence of the Kturn, as predicted in the present model. G172 and U173 were RNase sensitive, though they were predicted to be paired with C143 and G142 in the K-turn (Fig. 2). Other nucleotide residues, such as G52, C56, C57, C155, G164, and G165, which were predicted to participate in the formation of base pairs, were sensitive to digestion (Fig. 2). Because the secondary structure of PhopRNA was deduced by comparative sequence analysis, which presumably reflects the structure of PhopRNA in the fully assembled RNase P, the proposed

K-turn as well as helices P4 and P12.2 might be stabilized by interaction with the proteins, as discussed below. 3.4. SAXS analysis We have measured the SAXS profiles of PhopRNA to obtain the modeled structure by ab initio method using a scattering curve and several structural parameters, such as the radius of gyration (Rg), the maximum particle dimensions Dmax and the distance distribution function P(r). The Guinier plot and P(r) function obtained from the experimental scattering pattern are presented in Fig. 3A and B, respectively. The structural parameters of PhopRNA thus determined are given in Table 1, in comparison with those computed from the comparative 3-D model of PhopRNA as well as those of M1RNA obtained by SAXS analysis [35]. The structural parameters of PhopRNA are slightly smaller than those of M1RNA, consistent with its relative smaller size. The Rg and Dmax values derived from the SAXS measurements somewhat exceeds that of the comparative model and the scattering pattern and Kratky plot computed from the comparative model displays a slight deviations from the experimental SAXS data (Fig. 3A). The beads modeled structure of PhopRNA was reconstructed by the program DAMMIN using the SAXS experimental data, as described in Section 2 (Fig. 4A). The overlay between the comparatively obtained model and the low-resolution structure obtained from the scattering curve is shown in Fig. 4B. The comparative model appears similar but more compact than the low resolution shape of PhopRNA reconstructed ab initio by DAMMIN. As described above, the comparative 3-D model might reflect the structure of PhopRNA in the holoenzyme. It is thus suggested that PhopRNA might undergo a slight compactness upon complex formation with proteins that might represent the catalytically active conformation.

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Fig. 2. Enzymatic probing of PhopRNA. A, Primer extension analyses of RNA products using the primer P3. Lanes 1 and 2 indicate primer extension analyses of RNA products derived from the digested and undigested PhopRNA, as described in Section 2, respectively. Lanes G, C, A, and T are sequencing products using the same primer. Arrows indicate nucleotides at which the primer extension was stopped by enzymatic digestion. (B) Secondary structure of PhopRNA, summarizing the results of the enzymatic probing experiments on PhopRNA. The open arrows indicate phosphodiester bonds digested by RNases T1/A.

1

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Fig. 3. Small-angle X-ray scattering curves. (A) A comparison between a theoretical scattering curve of PhopRNA calculated using coordinates and a scattering profile obtained from experiment. Scattering curves calculated from coordinates (continuous line) and obtained from experiments using PhopRNA (open circle), whose intensities J(Q) were normalized using each forward scattering intensity, J(Q), were expressed on a Guinier plot. Inset: Kratky expression of calculated (continuous line) and experimental (open circle) scattering curves. (B) P(r) function from experimental data.

4. Discussion Table 1 Structural parameters for PhopRNA and M1RNA.

Rg (Å) Dmax (Å) a b

PhopRN A

3-D modela

M1RNAb

38.5 120

37.7 110

43.9 168–188

The comparative 3-D model in this study. The parameters were cited from Ref. [35].

In the present study, we reexamined the secondary structure of PhopRNA using stringent comparative tools to arrive at phylogenetically supported model. This model differs from previous versions derived from only a subset of sequences in that it includes a K-turn motif between helices P12.1 and P12.2, and several bulged residues (Supplementary Fig. 1). The presence of the K-turn motif in the PhopRNA had been predicted by chemical footprinting experiment which localized PhoRpp38 to PhopRNA helices P12.1

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Fig. 4. Model structures from SAXS ab initio method and comparative modeling. (A) Stereo view of the ab initio bead model of PhopRNA obtained by DAMMIM. (B) The overlayed SAXS ab initio model and the comparative 3-D model are illustrated as transparent sphere and ribbon cartoon, respectively. Figures were drawn with PyMol (http:// pymol.sourceforge.net).

and P12.2 comprised of nucleotides A116-G201 [15]. Because PhoRpp38 belongs to the L7Ae family of proteins known to recognize K-turns in 23S rRNA [27], the PhoRpp38 binding site was assumed to fold into the K-turn motif. The sequence analysis of helices P12.1 and P12.2 suggested that residues 141–148 and 170–174 have a potential to form the K-turn motif as a conserved feature of related archaeal RNase P RNA sequences (Supplementary Fig. 2). The K-turn motif was recently discovered also in the archaeal type M RNase P RNA from Methanococcus maripaludis [36] as well as eukaryotic RNase P and MRP RNAs [37]. Another PhoRpp38 binding site might involve residues G229–C276 of the PhopRNA helix P16 stem loop [15]. Examination of the PhopRNA sequence suggested a potential K-turn including 242-CCGAUGAG-249 and 259-UGAGG-263. This K-turn might mediate tertiary interaction involving helix P6 (Fig. 1A). This structure potentially forms in the RNase P RNAs of Methanothermus fervidus (Accession U42987), Methanopyrus kandleri (AE010305), two Pyrococcus species (AJ248283 and AE010137), three Thermococcus species (AF192364, AP006878, and AF192365), two Thermoplasma sequences (AL445066 and BA000011), as well as Picrophilus torridus (AE017261). Overall, the phylogenetic support for a K-turn at this location was poor, and other species closely related to P. horikoshii OT3 appeared to either contain a K-loop [38] or altogether lack this feature. Thus, this potential K-turn was not included in the PhopRNA model. The comparative modeling predicted that PhopRNA folds into a flattened disk-like structure with the approximate thickness 20 Å corresponding to one helix thick. The bacterial A-type RNase P RNA is made up of two layers [7,8]; the large layer of the structure contains most of the universally conserved regions, whereas the second layer comprises helical stems P13, P14, and P18, which are absent in the archaeal RNase P RNAs. It is known that tetraloop-helix interactions occur between P8 and L14 and P8 and

L18 in the bacterial A-type RNase P RNA [7,8]. These interactions ensure that the two domains (C- and S-domains) are positioned correctly to permit tRNA binding. Although the formation of the L9-P1 contact in archaeal RNase P RNAs has been reported to play an important role in orientation of the C- and S-domains [39], the lack for the second layer in PhopRNA may be compensated by the interaction with additional proteins. Indeed, it is known that PhoRpp38 binds to both stem-loops including helices P12.1/12.2 and P15/16 and thereby enhance the optimal temperature for pre-tRNA cleavage activity by the reconstituted particle [15]. Hence, this interaction may stabilize an appropriate conformation of the S- and C-domains. Furthermore, a recent mutational analysis suggests that the stem-loop including helix P8 in PhopRNA may serve as a binding site for the protein [40]. This interaction may also enforce the stabilization of a correct folding of the two domains. A detailed understanding of the molecular mechanism of archaeal RNase Ps requires knowledge of the high-resolution structure of the holoenzyme. However, progress in the structural determination of the holoenzyme has been slow due to two factors. The first is the difficulty in obtaining significant quantities of pure RNase P needed for crystallographic studies. The second factor is the complexity of the subunit components as compared to that of eubacterial RNase P. An alternative approach is to obtain high resolution structures of individual RNase P components by X-ray crystallography, and combined these data with knowledge of the overall arrangement of the components of the RNase P as analyzed by either SAXS or electron microscopy. Taking advantage of the intrinsic thermostability and a genetic property of the hyperthermophilic archaeon, the crystallographic analysis has established the structure of all five P. horikoshii RNase P protein components so far [16] and references therein. In the future, the 3-D model of PhopRNA presented here, together with existing and forthcoming

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crystallographic structural information of the other RNase P components will promote understanding of the structure and function of RNase P in much greater detail than is currently possible.

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Acknowledgments

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We thank Florian Müller for his generous support of the ERNA3D software package. This work was supported in part by Japan Society for the Promotion of Science Invitation Fellowship Program for Research in Japan (FY2008 to C.Z.) and by a grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan to M.K. (No. 22380062).

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Appendix A. Supplementary material

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Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2011.09.098.

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