The most complex RNA pseudouridylases are H/ACA ribonucleoprotein particles, which use a guide RNA for substrate capture and four proteins (Cbf5, Nop10, Gar1 and L7Ae/NHP2) for substrate modification. Here we report the three-dimensional structure of a catalytically deficient archaeal enzyme complex (including the guide RNA and three of the four essential proteins) bound to a substrate RNA. Extensive interactions of Cbf5 with one guide-substrate helix and a guide RNA stem shape the forked guide–substrate RNA complex structure and position the substrate in proximity of the Cbf5 catalytic center. Our structural and complementary fluorescence analyses also indicate that precise placement of the target uridine at the active site requires a conformation of the guide–substrate RNA duplex that is brought about by the previously identified concurrent interaction of the guide RNA with L7Ae and a composite Cbf5-Nop10 surface, and further identify a residue that is critical in this process.
Numerous noncoding RNAs that participate in nearly all aspects of gene expression and regulation are being discovered at a rapid rate1,2. Owing to their base-pairing potential, many noncoding RNAs act as guides in biochemical processes catalyzed by their partner proteins3. Ribonucleoprotein complexes guided by noncoding RNAs constitute an unconventional but widespread class of enzymes. One important class of these RNA-guided enzymes is the H/ACA small nucleolar (sno) RNPs that function in ribosome biogenesis in the nucleolus, isomerizing nearly 100 selected uridines to pseudouridines in human ribosomal RNA (rRNA)4,5,6. Many of these modified sites are within functionally critical regions of the ribosome7, and site-specific disruption of modifications impairs ribosome biogenesis or function8.
Since the role of H/ACA RNAs in rRNA pseudouridylation was first identified9,10, their known functional repertoire has expanded to include modification of small nuclear RNAs11 as well as rRNA processing12 and telomerase maturation13. H/ACA snoRNAs that guide rRNA modification are also found in the archaeal kingdom14, revealing the ancient evolutionary origin of H/ACA RNAs. Pseudouridylation contributes to RNA stability15,16,17,18, to increases in the number of available RNA functional groups19 and ultimately to the cellular function of the substrate RNA7,8,20,21,22,23,24.
The H/ACA RNAs responsible for isomerization of uridine function with partner proteins to form complex, multicomponent pseudouridylases (H/ACA RNPs)25,26. The H/ACA RNPs contain four core proteins that are known to participate directly in pseudouridylation27,28,29. These are Cbf5 (dyskerin in human, NAF57 in rodents), Gar1, Nop10 and L7Ae (NHP2 in eukaryotes). Cbf5 shares close sequence and structural homology with the bacterial pseudouridylase TruB and is the catalytic subunit for H/ACA RNP-mediated pseudouridylation30. Mutations in dyskerin cause the X-linked form of the rare genetic disease dyskeratosis congenita, which is characterized by abnormal skin pigmentation, bone marrow failure and an increased predisposition to cancer31,32. Archaeal H/ACA RNPs have been reconstituted from purified components and shown to possess pseudouridylation activity in vitro28,29. These studies showed that the ability of Cbf5 to catalyze efficient pseudouridylation is dependent upon each of the other three core H/ACA RNP components28,29. The specific roles of Gar1, Nop10 and L7Ae in pseudouridylation are not well understood.
In contrast to stand-alone pseudouridylases, which modify one or several specific uridine nucleotides33, H/ACA RNP pseudouridylases are capable of modifying as many different sites as are specified by the H/ACA guide RNAs expressed in the organism. The pseudouridylation guide H/ACA RNAs range in size from 70 to 250 nucleotides and are comprised of one to three helix-internal loop-helix-tail units2. Each unit is sufficient for binding all four protein factors and for guiding isomerization of a target uridine in vitro28,29,34. The strictly conserved 3′ tail (containing the ACA trinucleotide) and the asymmetric internal loop (pseudouridine pocket) are required for efficient binding of Cbf5 (refs. 28,29,34,35). The mechanistic details of how the target uridine of the substrate RNA is faithfully presented to the active site of Cbf5 are largely unknown. It is clear that the extensive base-pairing that occurs between the single-stranded substrate RNA and complementary nucleotides of the pseudouridine pocket of the H/ACA guide RNA determines the specificity for substrate RNAs9,10,35.
Recent structural studies of archaeal H/ACA RNPs have provided significant insight into the detailed architecture of the complex. Structures of isolated Cbf5–Nop10 and Cbf5–Nop10–Gar1 complexes have been determined36,37,38, and a structure of the complex containing all four proteins and a guide RNA is also available39. In addition, two structures of guide and target RNA complexes in solution were recently obtained40,41. These structures have all provided glimpses of the RNP enzyme. However, the basis for the accurate placement of target uridines in the active site of the enzyme, and for the requirement of all accessory proteins for function, is still not understood.
Toward the ultimate goal of understanding the molecular mechanism of H/ACA RNP function and the roles of the guide RNA and each protein in this process, we have determined the crystal structure of the Pyrococcus furiosus (Pf) H/ACA RNP bound to a wild-type substrate RNA for the Pf9 H/ACA guide RNA28 in the absence of L7Ae at 2.87 Å. To inhibit any potential chemical reaction during crystallization, we used a form of the Cbf5 protein containing an active-site mutation (D85A). The RNP structure revealed interactions between the guide–target RNA complex and conserved residues of the Cbf5 catalytic domain, and showed that guide–target RNA base-pairing in the context of Cbf5–Nop10–Gar1 complex places the target uridine in the vicinity of the active site of Cbf5. Structural comparison with the previously determined RNP structure in the absence of the substrate RNA but in the presence of L7Ae39 suggests a functional role of L7Ae in remodeling the guide RNA to further deliver the target uridine into the active site of Cbf5.
Overall structure of the RNP
The structure of the P. furiosus H/ACA RNP bound to a substrate RNA in the absence of L7Ae was determined by molecular replacement methods using the previously determined crystal structure of the P. furiosus Cbf5–Nop10–Gar1 complex (PDB accession code 2EY4) as the search model. Details of crystallization and the structural determination processes are described in Methods. The final structure was refined to an Rfree of 30.1%, including all reflections, and to a satisfactory stereochemical quality. The refined structural model contains residues 11–340 for Cbf5 (full-length 1–343), 3–55 for Nop10 (full-length 1–60), 1–73 for Gar1 (full-length 1–97), nucleotides 4–25 and 48–72 of the guide RNA (this represents all but the last three nucleotides of the bimolecular model guide RNA) and nucleotides 5–18 of the 21-mer unmodified target RNA (Fig. 1a). The predicted catalytic residue Asp85 in Cbf5 was mutated to alanine to inhibit potential pseudouridylation reactions during crystallization. For clarity, in what follows all guide RNA nucleotides are prefixed with 'g' and all target nucleotides are prefixed with 't'.
In this structure, the guide RNA is stably bound with the target RNA in the absence of the apical loop and kink-turn (Fig. 1a). The extended guide–target RNA helical complex adheres to an inclined plane primarily formed by the Cbf5 protein (Fig. 1b), which positions the target uridine close to the active site of Cbf5. The Cbf5-induced incline seems to be essential for positioning the substrate nucleotide, arguing for the importance of proteins in this process. Moreover, in this RNP structure, which lacks L7Ae, the target uridine is located ∼11 Å away from the active site of Cbf5 and is thus unavailable for modification. Previous in vitro reconstitution experiments had demonstrated a critical but unidentified role for L7Ae in pseudouridylation by the H/ACA RNP28,29. This RNP structure corroborates the biochemical data and furthermore indicates that L7Ae is also essential for target uridine placement within the active site of Cbf5.
Structural features of the guide–target RNA complex
As is also observed in the solution structures of the RNA–RNA complexes40,41, the target RNA is bound to the guide RNA through a constrained three-way junction (Fig. 1a,b). The upper stem of the guide RNA (P2) and the two helices formed between the 5′ and 3′ halves of the pseudouridine pocket and the substrate RNA (SH1 and SH2 for substrate helices 1 and 2) constitute the three branches of the junction, whereas the lower stem of the guide RNA (P1) restricts the direction of the branches (Fig. 1a,b). The pseudouridine pocket of the guide RNA is an elongated opening with a distance from the apex to the base of ∼40 Å (Fig. 2). The target RNA is bent into a V-shaped loop that fits snugly into the opening by base pairing to both sides of the pseudouridine pocket. The distance between the phosphate backbones of the two sides of the V-loop ranges from ∼6 Å to ∼14 Å (Fig. 2). SH1 and SH2 are each coaxial with the upper and lower stem of the guide RNA, P2 and P1, respectively (Fig. 1a,b), similar to what is seen in the guide–target RNA complexes formed in the absence of proteins and determined by NMR40,41. However, the two pseudocontinuous helices (P1-SH2 and P2-SH1) are inclined at a ∼120° angle at junction J2, in contrast to the nearly parallel helices in the guide–target RNA-only complexes40,41. This inclination, which appears to be a consequence of interaction with Cbf5, is necessary for the substrate RNA to enter the catalytic pocket, suggesting that Cbf5 has a functional role in the placement of the substrate RNA.
The guide–target RNA complex has an unprecedented RNA architecture wherein a single RNA strand docks to an asymmetric internal loop from one side. Unique to this architecture are the structural features observed at the helical junctions between P1 and SH1 (J1) and between SH2 and P2 (J2). J2, which has more complex features than J1, includes the target uridine and makes extensive contacts with Cbf5 residues (Fig. 3a). Both junctions involve rotation of downstream nucleotides away from the upstream helical procession. In J1, gA12 rotates away from SH2 helical stack to form an AU base pair with tU17 in the substrate RNA. This creates a sharp bend at the phosphate backbone between gA12 and gG11 (Fig. 2). Nucleotide rotations in J2 occur over nucleotides 52–54 in the guide as well as nucleotides 9–10 in the target strand. As a result of its extensive interactions with Cbf5 residues, the nucleobase of gC54 rotates toward the protein and away from tG9 (Fig. 3b,c). This eases the constraint on the phosphate backbone of tG9, allowing reversal of the target chain direction at this nucleotide; the phosphate backbone flips more than 90° away from the A-form position (Fig. 3b,c). The rotation at tG9 further disrupts the Watson-Crick edge-to-edge interaction between tG9 and gC54, leaving only one potential hydrogen bond between O6 of tG9 and N4 of gC54 (Fig. 3b,c). gU53 at the center of the J2 junction, which does not pair with a nucleotide, stabilizes the unpaired tG9 by stacking on its nucleobase (Fig. 3c). The sugar-phosphate of the target uridine, tU10, further facilitates the chain reversal, but its nucleobase is disordered in our structure. Finally, gU52 rotates nearly 90° from gU53 to pair with gG19 and initiate the P2 helix, completing the transition from SH2 to P2. The structure of J2 observed here is more open than that reported for the 3′ guide-target structure of the human U65 RNA40 in solution but appears to be less open than that of the 5′ guide-target structure of the same RNA in solution41. Whether these differences are due to the different RNA secondary structures or to the roles of the proteins remains to be explored. However, given that each of these RNAs guides the modification of its respective substrate by the same enzyme, it is likely that a similar structure is formed in the active site of the enzyme. Therefore, H/ACA RNPs could assemble a diverse range of guide–target RNA complexes to that required for catalysis. As discussed in greater detail below, phosphate backbone bending at the two junctions may also allow parts of the phosphate backbone to act as hinges for a structural transition required to dock the target RNA into the active site of the enzyme.
Interactions between the guide–target RNA complex and Cbf5
In the previously determined structure of the archaeal H/ACA RNP lacking target RNA, the pseudouridine pocket of the guide RNA is either disordered or stabilized by crystal packing interactions39. The primary contacts between the guide RNA and Cbf5 occur mostly in the lower stem of the RNA. Almost no interactions are observed between the pseudouridine pocket and the catalytic subunit of the pseudouridylase39. Our structure indicates that, upon association of the target RNA, the pseudouridine pocket of the guide RNA becomes ordered and forms specific contacts with conserved residues of Cbf5.
The most extensive contacts between the guide–target RNA complex and Cbf5 occur in the SH2 stem. As indicated by changes in the solvent-accessible surface areas attributable to the bound RNA, the Cbf5 residues that contact the SH2 stem are His63, Val66, Ala67, Ala68, Lys70, Gly79, His80, Thr100, Arg101, Val103, Gln104 and Lys325. Sequence alignments show that His63 and His80 are strictly conserved; homologous amino acids are found in the other positions in phylogenetically diverse organisms (Supplementary Fig. 1 online). Despite the extensive RNA-protein interface, few interactions involve nucleobases (Fig. 3a,b). This mode of interaction is consistent with the requirement that the pseudouridine pocket of the H/ACA RNA be available to interact specifically with the target RNA35,42,43. In contrast to the close interactions between SH2 and Cbf5, the SH1 stem formed between the 3′ half of the target RNA and the 5′ half of the pseudouridine pocket does not contact the proteins in our structure (Fig. 3b,c), which may contribute to the relatively high degree of disorder in this particular region of the RNA structure. In summary, the conservation of Cbf5 residues involved in stabilizing the pseudouridine pocket predicts the general importance of these residues in substrate binding and suggests that these interactions will be maintained in the fully assembled holoenzyme.
A mutation (S121G) in dyskerin (the human homolog of Cbf5) that affects an amino acid adjacent to the strictly conserved histidine that corresponds to Pf Cbf5 His80 (Supplementary Fig. 1) is associated with dyskeratosis congenita31. This is consistent with the previous suggestion that many dyskeratosis congenita–associated mutations affect amino acids in regions important for RNA binding37,38. Protein sequence alignment shows that the mutations underlying dyskeratosis congenita do not generally affect dyskerin amino acids that are predicted to be directly involved in RNA binding39 (which might cause a more severe phenotype), but rather neighboring amino acids (Supplementary Fig. 1). Notably, a mutation from an individual with dyskeratosis congenia was also recently mapped to human Nop10 (ref. 44) in a region expected to be involved in nonspecific binding the upper stem of the guide RNA in the holoenzyme39.
The conserved β7–β10 loop of Cbf5, which is predicted to interact with the guide–target RNA complex37,38,39, is disordered and not engaged in RNA-protein interactions in our structure (Fig. 4, dashed line). This loop may close the active site upon correct placement of the target RNA and the triggering mechanism may rely upon residues within the active site of the enzyme. The disengagement of the analogous 'thumb loop' of TruB upon mutation of the catalytic aspartate to alanine45 corroborates this possibility. Similarly, Gar1 may play a role in target RNA binding38,39, though it was not observed to interact with target RNA in our structure. Gar1 may also move closer to the target RNA when the target uridine is engaged in the active site.
Role of L7Ae in target RNA placement in the active site
Comparison of our Pf RNP structure with that of the Pf RNP containing L7Ae (and lacking target RNA) reveals substantial differences in the upper stem (P2) of the bound guide RNA. Structural and biochemical evidence described below suggests that these differences are attributable to L7Ae. We superimposed the Cbf5 molecules of both structures and examined the resulting positions of the other molecules. The overall structures of Cbf5, Gar1 and Nop10 are highly similar in both RNP structures (overall r.m.s. deviation 0.94 Å for 440 Cα atoms). In addition, the structures of the P1 lower stem, including the 3′ ACA trinucleotides of the guide RNAs, do not deviate substantially between the two (r.m.s. deviation 1.03 Å for 21 phosphate atoms). The largest differences between the two structures are in the pseudouridine pocket and the upper stem of the guide RNAs. Whereas the difference in the pseudouridine pockets is very likely the result of target binding as described above, that observed between the upper stems is likely the result of L7Ae binding. In the absence of L7Ae, the upper stem is less inclined forward toward Nop10 and more bent sideways in the direction of Gar1 (Fig. 4a). As a result, the contacts established between the upper stem and the protein complex are different (Fig. 4b). For instance, the upper stem extensively contacts residues 34–38 of Nop10 in the presence but not the absence of L7Ae (Fig. 4b). Similarly, the close contact formed between the upper stem of the guide RNA and Lys87 of Cbf5 in the RNP lacking L7Ae is not found in the RNP containing L7Ae (Fig. 4b). Notably, the results of protein protection assays indicate that full-length Pf9 also contacts Cbf5 Lys87 in the absence of both L7Ae and target RNA in solution46. These findings indicate that the difference in the position of the upper stem of the guide RNA in our structure does not reflect any alteration in the model guide RNA sequence or the presence of target RNA, but rather results from the lack of L7Ae.
L7Ae seems to bend the upper stem of the guide RNA away from the active site of Cbf5 and toward Nop10 (Fig. 4a). To specifically address what effect the L7Ae-induced movement of the upper stem might have on the proximity of the target uridine to the active site of Cbf5, we superimposed the upper stems (P2) of the guide RNAs from the two structures39 and allowed SH1 (which is not subject to extensive RNA-protein interaction in our structure) to follow while fixing the positions of P1 and SH2 (which are engaged in interaction with protein). Adjustments were restricted to the nucleotides in the two junction regions, J1 and J2, and were limited by steric constraints. The modeling produces an RNA configuration in which the target uridine is redirected toward the active site of Cbf5, to the position equivalent to that occupied by the target uridine in TruB-substrate RNA structures47,48 (Fig. 4c). The model requires unpairing of tG11 from gC18, and possibly pairing of gC18 with gU53 to 'close' the more open pocket observed in our structure and to more closely resemble the predicted consensus guide-target RNA structure35,42,43.
To investigate the proposed role of L7Ae in the target RNA placement, we devised a fluorescence assay that monitors the conformation of the nucleotide immediately 3′ of the target uridine (tG11). We replaced tG11 with the fluorescent analog of guanosine and adenosine, 2-aminopurine (2-AP), and the target uridine with 5-fluorouridine (5-FU). We believe that this target RNA mimics the rRNA substrate targeted by Pf9 RNA and that the 5-FU is converted by the enzyme to (5S,6R)-5-fluoro-6-hydroxypseudouridine (5-FhΨ), as has been observed in the case of the related TruB enzyme46. The fluorescence intensity of 2-AP directly reflects its stacking state and can, therefore, reveal changes in the conformational state of the RNA near the target uridine during the processes of substrate docking and the chemical reaction. The 2-AP–labeled target RNA was annealed with the guide RNA at a 1:1 molar ratio and then incubated with a ten-fold molar excess of Cbf5–Nop10–Gar1 trimeric protein complex to allow formation of an RNP similar to that observed in our crystal structure. In the absence of the L7Ae protein, the assembled H/ACA RNPs (either wild-type Cbf5 or the D85A mutant) showed a comparably low 2-AP fluorescence intensity, which we refer to as the 'low-fluorescence state' (Fig. 5). L7Ae was subsequently titrated into the RNP solution, which resulted in substantial increases in the 2-AP fluorescence intensity (Fig. 5), indicative of remodeling of the target RNA in the region of the target uridine. A maximum fluorescence intensity change was observed with a ten-fold molar excess of L7Ae. The change in 2-AP fluorescence required the presence of the Cbf5–Nop10–Gar1 trimeric protein complex because L7Ae titration of the guide RNA and target RNA in the absence of other proteins resulted in no change in fluorescence intensities (Fig. 5). We interpreted the L7Ae-induced change as a result of the bound 2-AP unstacking during the placement of the target RNA into the catalytic site of Cbf5 followed by conversion of 5-FU to 5-FhΨ. Consistent with this interpretation, when L7Ae was titrated into a solution of the 2-AP RNA bound with the trimeric protein bearing the D85A mutation in Cbf5 (the complex used for crystallization), the maximum fluorescence intensity change was substantially less than that produced by the wild-type RNP (Fig. 5), suggesting that this mutation prevents the target RNA from completing the L7Ae-induced transformation and/or the conversion of 5-FU to 5-FhΨ. We designate the final state reached by the wild-type RNP as the 'high-fluorescence state' and that reached by the mutant RNP as the 'intermediate-fluorescence state'. Thus, both the wild-type H/ACA RNP and the mutant H/ACA RNP lacking L7Ae were effectively shifted from the low-fluorescence state to another conformational state in an active site–dependent manner by L7Ae (Fig. 5).
The complexity of H/ACA RNP pseudouridylase assembly has been well documented27,28,29. Despite the close structural homology between Cbf5 and the stand-alone pseudouridylase TruB, in vitro activity assays using purified archaeal components have shown that three additional proteins are required for efficient modification by Cbf527,29. This knowledge has led to an intense and ongoing search for the molecular basis of the requirement for the accessory factors. As part of this effort, we report a crystal structure of the H/ACA RNP subcomplex containing Cbf5, Nop10, Gar1, an H/ACA RNA and a bound target RNA. Notably, we found that the guide RNA occupies a substantially different position in the absence of L7Ae than in an assembled apo H/ACA RNP structure39. The lack of L7Ae affects the locations of the upper stem and the pseudouridine pocket and thereby of the bound target RNA. Indeed, in this subcomplex the target uridine is found about 11 Å from the catalytic aspartate residue. On the basis of the assumption that the target RNA should be fully engaged in the active site in the holoenzyme, we propose that L7Ae has an important role in the placement of the target RNA. The concurrent interaction of L7Ae with the kink-turn motif of the guide RNA and with the composite surface formed by Nop10 and Cbf5 leads to anchoring of the upper stem of the guide RNA and consequently to functional docking of the target RNA (via the complementarity to the 5′ half of the pseudouridine pocket).
This proposal is supported by the results of our 2-AP fluorescence assay, which indicate that an L7Ae-induced conformational change occurs near the target uridine. In the absence of L7Ae, both the wild-type H/ACA RNP and that containing Cbf5 with an active-site mutation (D85A) stay in a low-fluorescence state that is likely to correspond to the crystal structure that we solved. L7Ae titration of both H/ACA RNPs resulted in marked increases in the fluorescence intensity, which is interpreted as a L7Ae-induced conformational change near the target uridine. However, we found that a complete L7Ae-induced conformational change (to the high-fluorescence state) requires the presence of the catalytic aspartate residue. One potential explanation for the sensitivity of the target RNA conformation to the catalytic aspartate is that conversion of 5-FU to 5-FhΨ causes an additional change in the conformation of the target uridine. Alternatively, a critical interaction established between the catalytic aspartate of the wild-type Cbf5 and the target uridine is required for the completion of the conformational change in the target RNA.
In this work, we have demonstrated that H/ACA RNP function requires remodeling of the guide RNA structure by L7Ae, assisted by Nop10, along with Cbf5 (and its catalytic residue), to place the target uridine at the active site for pseudouridylation.
Design and purification of the components used in crystallization.
To ensure that the D85A mutant of Cbf5 could bind Pf9 RNA, electrophoretic mobility shift assays were carried out as described28. These data indicated that the D85A mutant bound guide RNA with similar affinity as the wild-type protein (D. Baker, R.M.T. and M.P.T., unpublished data). We also formed crystals with the wild-type Cbf5 and the target RNA containing 5-fluorouridine and found that they were isomorphous to those containing the D85A mutant (Supplementary Table 1 online), suggesting that the mutant-containing complex has structural features grossly similar to those of the wild-type complex. Consistent with our observations, an aspartate-to-asparagine substitution in Cbf5's homolog, TruB, did not impair binding of its substrate RNA45. Proteins used in crystallization were purified according to published protocols38.
The model guide RNA used in crystallization contains a pseudouridine pocket identical to that of Pf9 but altered upper and lower stems, and it was assembled by hybridization of two RNA oligonucleotides. RNA oligos were purchased from Dharmacon and purified according to the manufacturer's recommended protocols.
The two guide strands and the target RNA, at a 1:1:1 molar ratio, were annealed by heating the solution for 1 min at 70 °C and then slowly cooling it. The RNA–protein complex was formed at a 1:1.2 molar ratio with a total concentration of 21.8 mg ml−1. The crystals were obtained by vapor diffusion methods in hanging drops at 30 °C. The RNA-protein mixture was mixed in an equal volume before being equilibrated with a reservoir solution containing 50 mM MES, pH 6.0, 100 mM NH4COOCH3, 5 mM MgSO4 and 1.0 M NaCl. Diamond-shaped crystals were obtained in 3–5 d. Crystals were soaked stepwise in cryosolutions containing the mother liquor plus 10% (v/v) and 15% (v/v) glycerol, respectively, before being flash cooled in a liquid nitrogen stream for data collection. Data were collected at the South Eastern Consortium Access Team (SER-CAT) beamline 22ID and were processed using HKL2000 (ref. 49). The crystals are in a primitive tetragonal space group with cell dimensions a = 96.56 Å, b = 96.56 Å, c = 240.98 Å. The solvent content of the crystals was determined to be 65.4%, which was consistent with the presence of one RNP in each asymmetric unit.
The structure was determined by molecular replacement methods using MOLREP50 through the CCP4i interface to the CCP4 programs51, which combines rotation and translation searches in a single step. The previously determined structure of the Cbf5–Nop10–Gar1 complex was used as a search model. A single and outstanding solution was found in the space group P41212. The trimeric protein complex transformed to the correct solution was subjected to successive rigid body, minimization and simulated annealing refinement. At this stage, SigmaA-weighted 3Fo − 2Fc and Fo − Fc electron densities were computed, which revealed the bound RNA molecules in the front surface of Cbf5–Nop10–Gar1 trimer. A molecular mask generated using a preliminary RNA model and the protein complex was then used to perform solvent flattening and flipping using initial phases computed from protein coordinates only in CNS52. The density-modified map was of sufficient quality (Supplementary Fig. 2 online) for tracing most RNA nucleotides. The real space correlation coefficients between the final model and the density modified map were 0.46 and 0.76 for RNA and proteins, respectively. Refinement of the RNP was carried out using CNS52 and REFMAC553. To account for rigid-body displacements of the complex with a reduced parameter set, the final stage of the refinement used translation-libration-screw-motion (TLS) parameters as implemented in REFMAC5. The three individual proteins and RNA strands were treated as single 'rigid-body' groups, and the final TLS parameters are listed in Supplementary Table 2 online. The refined structure has 0.45-Å coordinate error based on the maximum-likelihood method. A composite omit, cross-validated, SigmaA-weighted 3Fo − 2Fc map was computed using the final model and displayed around the RNA model (Fig. 2a) with 5% of the mode omitted at each time. The real space correlation coefficients between the final model and the composite omit 3Fo−2Fc map are 0.87 and 0.88 for proteins and RNA, respectively. The final protein structure was assessed with Procheck54 and found to be consistent with stereochemically valid models (Table 1).
Steady-state fluorescence studies.
The target RNA designed for fluorescence studies contains 2-AP at position tG11 and 5-FU at position tU10. The 2-AP– and 5-FU–labeled target RNA was purchased from Dharmacon. The Pf9 H/ACA guide RNA was obtained by T7 transcription. The annealed target–guide RNA complex at 1 μM concentration was then incubated with 10 μM of the Cbf5–Nop10–Gar1 or Cbf5(D85A)–Nop10–Gar1 protein complex in a total volume of 100 μl. L7Ae was titrated in small increments until its final concentration reached 10–15 μM, when fluorescence intensities no longer increased. Fluorescence measurements were performed in a Cary Eclipse fluorescence spectrophotometer (Varian). The sample cuvette was maintained at 50 °C using a circulating water bath. The excitation wavelength was 325 nm and the fluorescence intensity was measured at the peak of fluorescence (375 nm). The excitation and emission bandwidths were both 5 nm. For fluorescence intensity measurement after each titration, the sample was incubated for 20 min and then 8–10 accumulative scans were taken. Each titration was repeated in at least three independent experiments.
Protein Data Bank: Coordinates have been deposited with accession code 2RFK.
Protein Data Bank
Protein Data Bank
This work was supported by US National Institutes of Health (NIH) grant R01 GM66958-01 (H.L.) and NIH grant RO1 GM54682 (M.T. and R.T.). B. Liang is a predoctoral fellow of the American Heart Association, Florida/Puerto Rico Affiliate (0615182B). X-ray diffraction data were collected from the Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory. Supporting institutions for APS beamlines are listed at http://necat.chem.cornell.edu/ and http://www.ser-cat.org/members.html. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38.
Supplementary Figures 1 and 2 and Supplementary Tables 1 and 2
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Nature Structural & Molecular Biology (2009)