NMR structure of the active conformation of the Varkud satellite cleavage site

Bernd Hoffmann*, G. Thomas Mitchell*, Patrick Gendron†, Franc¸ois Major†, Angela A. Andersen‡, Richard A. Collins‡, and Pascale Legault*§

*Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602; †De´partement d’Informatique et de Recherche Ope´rationelle, Universite´de Montre´al, Montre´al, QC, Canada H3C 3J7; and ‡Department of Molecular and Medical Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8

Communicated by Ignacio Tinoco, University of California, Berkeley, CA, April 24, 2003 (received for review January 30, 2003) Substrate cleavage by the Neurospora Varkud satellite (VS) ri- The catalytic domain of the VS ribozyme recognizes its bozyme involves a structural change in the stem-loop I substrate stem-loop substrate primarily through tertiary interactions (7, 8). from an inactive to an active conformation. We have determined So far, the best-characterized tertiary interaction is a Mg2ϩ- the NMR solution structure of a mutant stem-loop I that mimics the dependent loop–loop interaction between stem-loops I and V, active conformation of the cleavage site internal loop. This struc- which is important for catalysis (Fig. 1a) (7, 11). Before tertiary ture shares many similarities, but also significant differences, with folding of the ribozyme, stem-loop I adopts an inactive confor- the previously determined structures of the inactive internal loop. mation, termed the unshifted helix (Fig. 1a) (11, 12). On binding The active internal loop displays different base-pairing interactions to stem-loop V, stem-loop I adopts an active conformation, and forms a novel RNA fold composed exclusively of sheared G-A termed the shifted helix (11, 12). In the active conformation of base pairs. From chemical-shift mapping we identified two Mg2؉ stem-loop I, base-pairing partners for G623, G624, and G625 are binding sites in the active internal loop. One of the Mg2؉ binding replaced by adjacent cytidines, creating a bulge at position 634 sites forms in the active but not the inactive conformation of the and reducing the size of the internal loop from 6 to 5 nt (Fig. 1a) internal loop and is likely important for catalysis. Using the struc- (11, 12). Stem-loop I variants that can form the active confor- ture comparison program MC-SEARCH, we identified the active in- mation are cleaved by the VS ribozyme; however, those that are ternal loop fold in other RNA structures. In Thermus thermophilus restricted to the inactive conformation are not (11). 16S rRNA, this RNA fold is directly involved in a long-range tertiary We report here the NMR structure of SL1Ј, a stem-loop I interaction. An analogous tertiary interaction may form between RNA that mimics the active conformation of the VS ribozyme the active internal loop of the substrate and the catalytic domain cleavage site internal loop (Fig. 1b). In addition, we present ϩ of the VS ribozyme. The combination of NMR and bioinformatic NMR chemical shift mapping of Mg2 binding sites in this RNA approaches presented here has identified a novel RNA fold and and a bioinformatic search of the SL1Ј internal loop fold in other provides insights into the structural basis of catalytic function in . This work reveals that the SL1Ј internal loop forms a the Neurospora VS ribozyme. novel RNA fold also present in 16S and 23S rRNA. Comparison between the active and inactive conformations (13, 14) of the NA molecules play essential roles in many cellular processes. substrate internal loop provides insights into the mechanism of RThese include the enzymatic activity of that are substrate activation by the VS ribozyme. required for protein synthesis and certain RNA processing Methods reactions (1). NMR and x-ray crystallographic studies have Sample Preparation. Unlabeled, 15N-labeled, and 13C͞15N-labeled provided some insights into the relationship between RNA Ј structure and catalysis; however, interpretation of structure– SL1 RNAs were synthesized in vitro by using T7 RNA poly- function relationships, even in the well studied hammerhead merase (generously provided by K. Morden, Louisiana State ribozyme (2), continues to be challenging (1). It has also been University, Baton Rouge), a synthetic oligonucleotide template (DNA Express, Macromolecular Resources, Fort Collins, CO), difficult to make any generalizations about the role of RNA Ј structure in catalysis, in part because of the small number of and nucleoside triphosphates (15, 16). The 23-mer SL1 was purified by denaturing gel electrophoresis, dephosphorylated at known ribozymes and the limited amount of structural infor- Ј mation available. We are studying the Neurospora Varkud its 5 end with calf alkaline phosphatase (Roche Molecular satellite (VS) ribozyme to provide information about the role of Biochemicals), and further purified by DEAE-Sephacel chro- tertiary structure and conformational changes in RNA catalysis. matography (Amersham Biosciences). The RNA (1–5 mM) was exchanged into 10 mM d11-Tris (pH 7.0), 50 mM NaCl, 0.2 mM The Neurospora VS ribozyme originates from an abundant ͞ RNA satellite of 881 nt found in the mitochondria of the EDTA, 0.05 mM NaN3 in 9:1 H2O D2OorD2O. Before each set Varkud-1c strain of Neurospora (3). Fragments of Ϸ120–180 nt of NMR experiments the RNA was heated to 95°C for 2 min and snap-cooled in ice water. derived from this natural RNA sequence undergo self-cleavage at a specific phosphodiester bond to produce 5Ј-OH and 2Ј,3Ј- NMR Spectroscopy. NMR data were collected at 25°C on a Varian cyclic phosphate termini (Fig. 1a) (3–5). Although these prod- Inova 600-MHz spectrometer equipped with a z-axis pulse-field ucts are characteristic of other small ribozymes, the VS ribozyme gradient probe, either a 1H{13C͞15N} triple resonance probe or possesses unique primary (4), secondary (6), and tertiary struc- a 1H-19F{15N-31P} indirect detection probe. 1H, 13C, and 15N tures (7–9). The secondary structure of the self-cleaving VS chemical shift assignments were obtained by using the following ribozyme is characterized by six helical domains (Fig. 1a); experiments: 2D 1H-15N heteronuclear single quantum coher- stem-loop I forms the substrate domain and stem-loops II–VI comprise the catalytic domain (6). When these two domains are synthesized separately, the catalytic domain can perform the Abbreviations: HSQC, heteronuclear single quantum coherence; NOE, nuclear Overhauser same cleavage reaction, known as the trans reaction, with effect; PDB, Protein Data Bank; rmsd, rms deviation; VS, Varkud satellite. multiple turnover (10). There is no reported high-resolution Data deposition: The atomic coordinates have been deposited in the Protein Data Bank,

structure of the full VS ribozyme at this time, but tertiary www.rcsb.org (PDB ID code 1OW9). BIOCHEMISTRY structure models have been proposed (8, 9). §To whom correspondence should be addressed. E-mail: [email protected].

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0832440100 PNAS ͉ June 10, 2003 ͉ vol. 100 ͉ no. 12 ͉ 7003–7008 Downloaded by guest on September 28, 2021 Fig. 1. (a) Sequence and secondary structure of the Neurospora VS ribozyme. The interaction between stem-loops I and V is indicated by a dashed line, and residues involved in this interaction are circled (7). Upon tertiary folding of the ribozyme, stem-loop I (subdivided into Ia and Ib) undergoes a structural change from an inactive to an active conformation (11). The minimal substrate domain for the trans cleavage reaction is boxed (10), and the cleavage site is indicated by the arrowhead. (b) Sequence and secondary structure of SL1Ј RNA. WT and mutant are represented by uppercase and lowercase letters, respectively. (c) SL1Ј is cleaved by the VS ribozyme. 3Ј-32P-end-labeled SL1Ј (25 nM) was incubated at 30°C in 40 mM Tris⅐HCl (pH 8.0), 50 mM KCl, and 100 mM MgCl2 in the presence (lanes 1 and 2) or absence (lanes 3 and 4) of unlabeled RZ6 ribozyme (51) (37 ␮M) for 0 h (lanes 1 and 3) or 16 h (lanes 2 and 4) (12). Full-length SL1Ј and the 3Ј product (3Ј-P) were separated by denaturing PAGE and visualized with a PhosphorImager.

ence (HSQC), 2D 1H-1H flip-back watergate-NOESY, 2D G- 2͞5 proton and carbon atoms of SL1Ј. The chemical-shift specific H(NC)-total correlation spectroscopy (TOCSY)-(C)H, changes (⌬ in ppm Ϯ 0.03 ppm) are calculated according to the ⌬ϭ ⌬ 2 ϩ ϫ⌬ 2 1͞2 ⌬ ⌬ 2D A-specific (H)N(C)-TOCSY-(C)H, 2D C-specific and U- equation [( H) (0.3 C) ] , where H and C are, specific H(NCCC)H, 2D 1H-13C constant-time HSQC, 2D 1H- respectively, the 1H and 13C chemical-shift differences between 15N MQ-(HC)N(C)H, 3D HCCH-COSY, 3D HCCH-TOCSY, the assigned 1H-13C constant-time HSQC (26) correlations of 13 ͞ ͞ and 3D C-edited heteronuclear multiple quantum correlation- the control sample [10 mM d11-Tris (pH 7.0) 50 mM NaCl 0.05 2ϩ NOESY (17). Distance restraints were derived from nuclear mM NaN3] and those of the Mg -supplemented sample. The 13 ͞15 ⌬ ⌬ Ј Ј Overhauser effect (NOE) in 3D C N-edited NOESY-HSQC values of H and C were also tabulated for the resolved 1 ,2, (80 and 240 ms) (18), 3D 13C-edited heteronuclear multiple 3Ј,6͞8, and 2͞5 protons and carbons after each addition of 1 15 quantum correlation-NOESY (80 and 240 ms), and 2D H- N MgCl2 (0.25, 0.5, 0.75, 1.0, 2.0, 5.0, 10, 20, and 40 mM MgCl2). Carr Purcell Meiboom Gill-NOESY (150 ms) (19) experiments. These data were used to obtain apparent Kd values when the total ⌬ Ն Ն 2D HNN-COSY (20) and 2D H(CN)N(H) (21) spectra were change at 40 mM MgCl2 ( T) was 0.1 ppm and 0.45 ppm for 2 1 13 collected to detect JNN couplings across hydrogen bonds in H and C chemical shifts, respectively. Apparent Kd values were Watson–Crick and sheared G-A base pairs, respectively. A 2D calculated by assuming single independent 1:1 binding models by ␦ ⌬ ϭ ⌬ ͞ ϫ ϫ ϩ double quantum filtered COSY was collected to define torsion fitting the equation obs ( T) (2 [RNA]T) {(M [RNA]T ͞ ϩ Ϫ ϩ ϩ 2 Ϫ ϫ 1͞2 angles. Spectra were processed with NMRPIPE NMRDRAW (22) Kd) ((M [RNA]T Kd) (4 M [RNA]T)) } (27, 28), ⌬ and analyzed with NMRVIEW (23). where obs is the observed shift at each MgCl2 concentration and [RNA]T and M are the total concentrations of RNA and metal Structure Calculations. NOE-derived distance restraints were sep- ion, respectively. Because multiple binding sites are detected in Ј arated in four ranges, strong (1.8–3.3 Å), medium (1.8–3.9 Å), SL1 , the apparent Kd represents an upper value of the real Kd weak (1.8–5.5 Å), or very weak (1.8–7.0 Å). Distance ranges (27, 28). with lower bounds of 2.5–4.0 Å and upper bounds of 40 Å were defined for very small or absent NOESY crosspeaks between Pattern Search of RNA Structures in the Protein Data Bank (PDB). H1Ј,H2Ј, and H6͞H8 protons of residue i and H5Ј͞H5ЈЈ protons Using MC-SEARCH (P.G. and F.M., unpublished data), we of residues i, i Ϫ 1, and i ϩ 1. Because of strong NMR evidence searched for other occurrences of the SL1Ј internal loop fold. for formation of Watson–Crick base pairs in helices Ia and Ib and MC-SEARCH is a computer program, derived from the automated of the sheared G-A base pair in the tetraloop, canonical distance RNA annotation program MC-ANNOTATE (29, 30), that searches restraints were used to define these base pairs. In the internal RNA structure files for regions that match a user-defined loop, G5 N2–A19 N7 and G18 N2–A6 N7 distances were pattern. The searched pattern was described only in terms of restrained to 1.8–3.15 Å based on results from the 2D H(C- primary and secondary structures; the sequence of the residues N)N(H) spectrum (21). Torsion angle constraints were derived in the internal loop was fixed to that of the SL1Ј internal loop, from a 2D double quantum filtered COSY (for ␦) and compar- but the sequence of the closing base pairs was not fixed (30). No ative analyses of NOE data (for ␦ and ␥). additional structural interactions were included in the search 3D structures were calculated with restrained molecular dy- pattern for the bases in the internal loop. The searched database namics and simulated annealing in X-PLOR 3.840 (24). From a set contained high-resolution crystal structures (3.0 Å or less) of the of 50 structures with randomized torsion angles, 29 structures PDB (31). The identified structures were compared and classi- satisfied the experimental restraints (no distance violation Ͼ0.1 fied by using hierarchical clustering based on the rms deviation Å and no torsion angle violation Ͼ5°), and, from these, the 10 (rmsd) metric (29). lowest energy structures were selected for analysis. An average structure was calculated from the 29 final structures and mini- Results and Discussion mized against the experimental restraints. The program MOLMOL Structure Determination. To study the active form of the stem-loop was used for visualization and structural analysis (25). I internal loop, we initially synthesized a series of mutant stem-loop I RNAs with sequences designed to form only the Metal Binding Studies. Chemical-shift changes induced by addition active conformation (11, 12). All of the RNAs tested that retain Ј Ј Ј ͞ of 10 mM MgCl2 were monitored for all of the 1 ,2,3,6 8, and the natural sequence in the terminal loop I did not adopt a single

7004 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0832440100 Hoffmann et al. Downloaded by guest on September 28, 2021 Table 1. Structural statistics Description of the Active Conformation of the Stem-Loop I Internal Loop. Distance restraints The internal loop of the active stem-loop I adopts a novel From standard NOESY 1,081 structural fold, which is composed entirely of sheared G-A base Internucleotide 608 pairs (Fig. 2). The geometries of all G-A base pairs in the internal Intranucleotide 473 loop are defined by many experimental restraints, including From 2D 1H-15N Carr Purcell Meiboom Gill-NOESY 24 NOEs from exchange-broadened amino groups (Fig. 6a, which Hydrogen bonds from 2J couplings 9 is published as supporting information on the PNAS web site, NN 2 Others (W-C and G11-A14) 37 www.pnas.org) (19) and JNN couplings across the GN2-AN7 rmsd from experimental restraints hydrogen bond (Fig. 6b) (21). Theoretical distance or planarity Distance restraints, Å (1,105) 0.0068 Ϯ 0.0004 restraints were not used to define base-pairing in the internal Dihedral restraints, ° (40) 0.13 Ϯ 0.02 loop. Residues G620 and A639 adopt a canonical sheared G-A rmsd from idealized geometry base pair, with planarity of the two bases (Fig. 2c). The most Bonds, Å 0.00452 Ϯ 0.00003 striking feature of this internal loop is the sharing of G638 for the Angles, ° 1.036 Ϯ 0.003 formation of two sheared G-A base pairs, G638–A621 and Impropers, ° 0.361 Ϯ 0.004 G638–A622 (Fig. 2). We will refer to this structural element as Heavy-atom rmsd, Å the shared sheared G-A base pairs. In all of the calculated Overall (residues 2–22) 1.00 Ϯ 0.44 structures, the base of G638 is not coplanar with the base of GAAA tetraloop (residues 10–15) 0.21 Ϯ 0.08 either A621 or A622, but rather lies between the two planes Internal loop (residues 5–7 and 18–19) 0.27 Ϯ 0.13 defined by these adenines (Fig. 2). We observed short interresi- due distances characteristic of either direct or water-mediated hydrogen bonds for both G-A base pairs (Fig. 2c): G638 N3– A621 H62 (2.41–2.63 Å); G638 H22–A621 N7 (2.28–2.84 Å); conformation and were not suitable for further NMR analysis. G638 N3–A622 H62 (2.55–3.25 Å); and G638 H22–A622 N7 Only the RNA shown in Fig. 1b gave high-quality NMR data. Ј (2.97–3.30 Å for 9 of 10 structures). There is no evidence for slow This stem-loop I mimic, termed SL1 , contains WT nucleotides dynamics in the internal loop; however, the possibility for fast for the active internal loop and for at least one closing base pair Ј dynamic averaging (time scale less than milliseconds) between on each side of the internal loop (Fig. 1b). SL1 is cleaved in the multiple base-pairing schemes cannot be ruled out. presence but not the absence of the catalytic domain of the VS Ј ribozyme (Fig. 1c). As expected, cleavage of SL1 is slower than Comparison with the Inactive Conformation of the Stem-Loop I Ј for WT stem-loop I, because SL1 cannot form the loop–loop Internal Loop. Two structures of the inactive form of the stem- interaction with stem-loop V (11). loop I internal loop had been previously determined by NMR Ј The structure of SL1 was determined by using heteronuclear spectroscopy (13, 14). In both cases, the inactive internal loop is NMR methods, and 3D structures were calculated by using characterized by tandem sheared G-A base pairs and a wobble ϩ Ϸ restrained molecular dynamics and simulated annealing. The A622 –C637 base pair with a pKa of 6.2 for the adenine imino structural statistics (Table 1) and the superposition of the 10 group (Fig. 3a) (13, 14). From pH-dependent 15N chemical-shift Ϯ lowest energy structures (Fig. 2a) indicate that the structure of studies, a pKa of 4.0 ( 0.1) was determined for A622 in the this RNA, particularly of the active internal loop, is well defined active internal loop (not shown). Although the deprotonation of by the NMR data. The individual stems and the GAAA tetra- A622ϩ may help to allow the transition to the active conforma- loop are also individually well defined and adopt A-form helix tion, it is not sufficient because the interaction with stem-loop V and GNRA tetraloop conformations (32), respectively. is also required (11, 12).

Fig. 2. NMR structure of SL1Ј.(a) Stereoview of the heavy-atom superposition of the 10 lowest energy structures. (b) Minimized average structure of the active internal loop viewed from the minor groove (Upper) and down the helix axis (Lower). The phosphorus at the cleavage site is shown as a yellow sphere. (c) Base pair geometry for internal loop residues in the minimized average structure. The 2Ј-OH and amino protons are shown here in addition to the heavy atoms. The

dashed lines represent potential hydrogen bonds based on short distances (Ͻ3.4 Å) observed in at least one structure. Internal loop nucleotides are colored BIOCHEMISTRY according to the scheme presented in b.

Hoffmann et al. PNAS ͉ June 10, 2003 ͉ vol. 100 ͉ no. 12 ͉ 7005 Downloaded by guest on September 28, 2021 Fig. 3. Structural comparison of the inactive and active conformations of the VS ribozyme stem-loop I internal loop. (a) Summary of the base-pairing interactions in the inactive (Left) (13, 14) and active (Right) conformations. (b) Stereoview for comparing the inactive and active conformations. The inactive stem-loop I conformation (pastel colors) is the best representative conformer from the ensemble of NMR structures of Michiels et al. (ref. 13; PDB ID code 1E4P), and the active conformation (darker colors) is the minimized average structure described here. The superposition was obtained by minimizing the rmsd for heavy atoms of residues C619–G623 and C637–G640. Internal loop nucleotides are colored according to the scheme presented in a.

We superimposed heavy atoms of residues C619–G623 and C637–G640 of the minimized average structure of SL1Ј with those of the inactive internal loop structure of Michiels et al. (13) (Fig. 3b). The heavy atom rmsd for this superposition is 2.0 Å. Overall, there are many similarities between these two struc- tures, including the sheared G620–A639 and G638–A621 base Fig. 4. Mg2ϩ binding sites of SL1Ј.(a) Schematic comparing the 5Ј-A G-3Ј͞5Ј-C pairs, as well as the cross-strand stacking between G620 and G-3Ј metal-ion binding motif (box) of the (Left) (36) G638 and between A621 and A639 (Figs. 2b and 3b). The largest and the two 5Ј-A G-3Ј͞5Ј-C G-3Ј motifs (boxes) in the internal loop of SL1Ј differences between the two structures are at one end of the (Right). For the hammerhead ribozyme, the bound Mg2ϩ is represented by a internal loop, near residues A622 and C637. In the inactive form, red circle, and the cleavage site is indicated by the arrowhead. (b) Chemical- ϩ the protonated A622 forms a wobble A622 –C637 base pair, shift changes (⌬ in ppm Ϯ 0.03 ppm; see Methods) after addition of 10 mM Ј Ј Ј ͞ ͞ Ј whereas in the active form the deprotonated A622 forms a MgCl2 for the 1 ,2,3,68, and 2 5 proton and carbon atoms of all SL1 2ϩ sheared G-A base pair with G638, and C637 forms a Watson– residues. (c) Summary of Mg binding data on the stereoview of the mini- Ј Crick G-C base pair with G623. These differences in base-pairing mized average structure of SL1 (residues C4-A9 and U16-G20). Significant chemical-shift changes after addition of 10 mM MgCl2 (⌬Ͼ0.2 ppm) were affect the position of the phosphate backbone for residues A621, mapped as green spheres on the respective C1Ј,C2Ј,C3Ј,C6͞C8, and C2͞C5 A622, and G638 (Fig. 3b) and the positions of stem Ib residues atoms. Putative locations for the Mg2ϩ (red spheres) were obtained by heavy- with respect to the internal loop (not shown). Despite these atom superposition of the 5Ј-A G-3Ј͞5Ј-C G-3Ј motif from the x-ray structure seemingly subtle conformational differences, the active stem- of the Mg2ϩ-bound hammerhead ribozyme (only Mg2ϩ are shown) (36) with loop I is effectively cleaved by the catalytic domain of the VS the 5Ј-A639 G640-3Ј͞5Ј-C619 G 620-3Ј (rmsd of 1.01 Å) and the 5Ј-A622 ribozyme, whereas stem-loop I mutants locked in the inactive G623-3Ј͞5Ј-C637 G 638-3Ј (rmsd of 1.56 Å) motifs from the minimized average Ј conformation are not (11). As will be presented below, the active structure of SL1 . internal loop conformation provides two unique structural char- acteristics: a divalent metal-ion binding site and a tertiary interaction motif. biological cation necessary for catalysis (33). From the NMR structure of SL1Ј, we find that the active internal loop contains Interactions of SL1؅ with Mg2؉. Although cleavage by the VS two 5Ј-A R-3Ј͞5Ј-Y G-3Ј motifs previously identified as divalent ribozyme has been detected with several divalent metal ions (33) metal-ion binding sites in other RNAs (Fig. 4a) (35–37). In the 2ϩ 2ϩ and monovalent salts (2 M Li2SO4) (34), Mg is likely the crystal structure of the hammerhead ribozyme, a Mg is found

7006 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0832440100 Hoffmann et al. Downloaded by guest on September 28, 2021 Fig. 5. The internal loop structure of the active stem-loop I is a tertiary interaction motif. (a) Sequence and secondary structure of the pattern searched by MC-SEARCH and of helix 44 of 16S rRNA of T. thermophilus (40). (b) A superposition between helix 44 (PDB ID code 1FJG; pastel colors) and the minimized average structure of SL1Ј (darker colors) was obtained by minimizing the rmsd for heavy atoms of the five residues in the internal loop (1.40 Å). Also shown are residues in helix 13 of 16S rRNA, which form a tertiary interaction with helix 44. (c) Summary of the base-pairing and stacking interactions in helices 13 and 44 and of tertiary contacts between them. Solid and dashed black lines indicate base pairs with two hydrogen bonds (either Watson–Crick or sheared G-A) and one hydrogen bond, respectively. Black rectangles indicate base stacking. Red spheres indicate riboses involved in ribose–ribose contacts (red dashed lines). The two adenines A1433 and A1434 participate in A-minor motifs (green dashed lines).

associated to a 5Ј-A G-3Ј͞5Ј-C G-3Ј motif, within close distance of potential Mg2ϩ ligands at this site impaired substrate cleavage of the A 5Ј phosphate and of the G N7 on the 5Ј-A G-3Ј strand (11, 38), it is likely that this Mg2ϩ plays an important role in (Fig. 4a) (36). The importance of the internal loop 5Ј-A622 catalysis. Interestingly, modeling of a Mg2ϩ at the 5Ј-A622 G623-3Ј͞5Ј-C637 G638-3Ј motif for catalysis by the VS ribozyme G623-3Ј͞5Ј-C637 G638-3Ј motif positions this ion in the vicinity is supported by biochemical data. Mutational studies have of the phosphate at the cleavage site (Fig. 4c). This Mg2ϩ may indicated that the 623–637 base pair must fit the R-Y consensus be important for substrate docking in the active site or the (11), and phosphorothioate substitution experiments have dem- chemical reactions of the VS ribozyme. onstrated the importance of the 5Ј phosphate of A622 for catalysis (38). Other Occurrences of the Active Internal Loop Fold. To investigate We used chemical-shift mapping to investigate the Mg2ϩ whether the SL1Ј internal loop fold exists in other RNAs, we binding sites in SL1Ј.2D1H-13C constant-time HSQC spectra of used an automated program MC-SEARCH (P.G. and F.M., un- SL1Ј were recorded before and after addition of various con- published data) to search for RNA structure patterns in the PDB Ј centrations of MgCl2. After addition of 10 mM MgCl2, signifi- (31). The pattern searched contained the sequence of the SL1 cant changes in 1H and 13C chemical shifts (⌬Ͼ0.2 ppm) are internal loop flanked by closing base pairs (Fig. 5a). MC-SEARCH observed at the end of the helix, in the internal loop, and in the found several occurrences of this pattern (Fig. 5a) in the GAAA tetraloop (Fig. 4b). These changes are mapped onto the database, consisting of multiple structures of two different Ј 2ϩ structure of SL1 in Fig. 4c. Apparent Kd values for Mg were helical domains of rRNA. One is from helix 44 of 16S rRNA calculated based on Mg2ϩ-dependent 1H and 13C chemical-shift found in the small ribosomal subunit of Thermus thermophilus changes for resolved signals (Fig. 7, which is published as (40) (Fig. 5). The other one is from helix 25 of 23S rRNA found supporting information on the PNAS web site) (27). For the in the large ribosomal subunit of Haloarcula marismortui (41) Ϯ GAAA tetraloop, a Kd of 3.4 0.6 mM was obtained, in (Fig. 8, which is published as supporting information on the agreement with published data (28, 39). In the internal loop, two PNAS web site). Surprisingly, even though no hydrogen bonding 2ϩ Ϯ regions are affected by Mg ; we obtained a Kd of 3.6 0.7 mM or stacking interactions were specified in the internal loop of the Ϯ for residues A621, A622, and G623 and a Kd of 2.4 1.1 mM for pattern searched, the RNAs found are very similar in structure residues A639 and G640. These results suggest that there are at to the internal loop of SL1Ј. The sequence of helix 44 of 16S least two Mg2ϩ binding sites in the active internal loop and that rRNA matches exactly the sequence of the internal loop of SL1Ј each Mg2ϩ binding site is located near the 5Ј-A G-3Ј strand of and its closing base pairs (Fig. 5a). Heavy-atom superposition of a5Ј-A G-3Ј͞5Ј-C G-3Ј motif. We attempted to locate more internal loop residues of helix 44 of 16S rRNA [PDB ID code precisely the divalent metal binding sites in SL1Ј by using cobalt 1FJG (40)] and SL1Ј yields a rmsd of 1.40 Å (Fig. 5b). The main hexammine and MnCl2. However, because of the presence of difference between these structures is in the shared sheared G-A multiple binding sites, the data were too complex and could not base pairs; in helix 44, A1433 is displaced away from G1467 such be interpreted unambiguously. To illustrate the possible location that there is only one potential hydrogen bond between its base of the Mg2ϩ binding sites in the internal loop of the VS ribozyme, and the base of G1467 (Fig. 5). we superimposed the Mg2ϩ-bound 5Ј-A G-3Ј͞5Ј-C G-3Ј motif of Interestingly, the minor grooves of helix 44 of 16S rRNA and the hammerhead crystal structure with each of these two 5Ј-A helix 25 of 23S rRNA both participate in long-range tertiary G-3Ј͞5Ј-C G-3Ј motifs of SL1Ј (Fig. 4c). interactions termed canonical ribose zippers (Figs. 5 and 8) (42). The 5Ј-A639 G640-3Ј͞5Ј-C619 G620-3Ј structural motif, In 16S rRNA of T. thermophilus, the internal loop of helix 44 present in both the inactive and active internal loops, is not part interacts with the stem of helix 13 through multiple ribose–ribose of the minimal stem-loop I substrate and is therefore not contacts, and the two adenines, A1433 and A1434, participate in essential for catalysis (Fig. 1a). The 5Ј-A622 G623-3Ј͞5Ј-C637 A-minor motifs with C335 and G319-C334, respectively (Fig. 5c) G638-3Ј motif, however, is found solely in the active internal loop (43). This ribose zipper is similar to that formed by the GAAA and is essential for activity (11, 38). Our chemical-shift mapping tetraloop and tetraloop receptor, where two consecutive ad- ϩ data indicate that a Mg2 binds near the 5Ј-A622 G623-3Ј site of enines stacked on a sheared G-A base pair participate in BIOCHEMISTRY the internal loop. Because mutations and chemical modifications A-minor motifs (42, 44, 45). In addition, the base stacking

Hoffmann et al. PNAS ͉ June 10, 2003 ͉ vol. 100 ͉ no. 12 ͉ 7007 Downloaded by guest on September 28, 2021 pattern in helix 44 of 16S rRNA (43) and in the internal loop of Conclusion Ј SL1 is strikingly similar to that of the GNRA fold (32). The NMR study of the active internal loop of the VS ribozyme For optimal cleavage by the VS ribozyme, the stem-loop I reveals a novel RNA fold. This fold consists exclusively of sheared substrate must participate in multiple tertiary interactions with G-A base pairs and forms only in the active but not the inactive the catalytic domain (7, 8). These include the well characterized conformation of the VS ribozyme substrate internal loop. The loop–loop interaction with stem-loop V (Fig. 1a) (7) and inter- actions with helix II and helix VI (8, 46, 47), which contains the active internal loop conformation differs from the inactive one by proposed active site (38, 46, 48–50). Substrate binding to the the rearrangement of base pairs and formation of a divalent catalytic domain of the VS ribozyme is facilitated by formation metal-ion binding site that appears important for catalysis. In of the active stem-loop I conformation (8, 51). Here, we have addition, a bioinformatic analysis has identified the active internal shown that this active conformation functions as a tertiary ribose loop fold as part of tertiary interaction motifs in rRNAs. Future zipper motif in 16S and 23S rRNAs (Fig. 5). Analysis of structural studies will determine whether a similar tertiary inter- stem-loop ribose zippers in rRNA structures indicates that action forms in the Neurospora VS ribozyme. adenines are favored in the loop (42); a similar preference for adenine residues at positions 621 and 622 in the stem-loop I of We thank J. G. Omichinski for discussions, K. Morden for T7 RNA the VS ribozyme has been demonstrated from in vitro selection polymerase, A. Majumdar for pulse sequences, and W. G. Scott for PDB experiments (11). In the active stem-loop I conformation, A621 coordinates of the hammerhead ground-state structure with Mg2ϩ. This and A622 are well positioned to form A-minor motifs (Fig. 5b), ϩ work was supported in part by a Basil O’Connor Starter Scholar whereas in the inactive stem-loop I, the A622 –C637 base pair Research Award from the March of Dimes Birth Defects Foundation and (Fig. 3) would hinder such interaction. Based on these similar- a National Science Foundation Career Award (to P.L.), the Canadian ities in sequence and structure, we speculate that the active Institutes of Health Research (R.A.C. and F.M.), and the National conformation of the VS ribozyme stem-loop I internal loop Science and Engineering Research Council (F.M.). R.A.C. is a Canada forms a ribose zipper with either helix II or VI of the catalytic Research Chair, and F.M. is a Canadian Institutes of Health Research domain. Investigator.

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