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Solution structure of a GAAA tetraloop receptor RNA

1997; Springer Nature; Volume: 16; Issue: 24 Linguagem: Inglês

10.1093/emboj/16.24.7490

1460-2075

Samuel Butcher, Thorsten Dieckmann, Juli Feigon,

RNA Research and Splicing

Article15 December 1997free access Solution structure of a GAAA tetraloop receptor RNA Samuel E. Butcher Samuel E. Butcher Department of Chemistry and Biochemistry, and Molecular Biology Institute, University of California, Los Angeles, CA, 90095-1569 USA Search for more papers by this author Thorsten Dieckmann Thorsten Dieckmann Department of Chemistry and Biochemistry, and Molecular Biology Institute, University of California, Los Angeles, CA, 90095-1569 USA Search for more papers by this author Juli Feigon Corresponding Author Juli Feigon Department of Chemistry and Biochemistry, and Molecular Biology Institute, University of California, Los Angeles, CA, 90095-1569 USA Search for more papers by this author Samuel E. Butcher Samuel E. Butcher Department of Chemistry and Biochemistry, and Molecular Biology Institute, University of California, Los Angeles, CA, 90095-1569 USA Search for more papers by this author Thorsten Dieckmann Thorsten Dieckmann Department of Chemistry and Biochemistry, and Molecular Biology Institute, University of California, Los Angeles, CA, 90095-1569 USA Search for more papers by this author Juli Feigon Corresponding Author Juli Feigon Department of Chemistry and Biochemistry, and Molecular Biology Institute, University of California, Los Angeles, CA, 90095-1569 USA Search for more papers by this author Author Information Samuel E. Butcher1, Thorsten Dieckmann1 and Juli Feigon 1 1Department of Chemistry and Biochemistry, and Molecular Biology Institute, University of California, Los Angeles, CA, 90095-1569 USA The EMBO Journal (1997)16:7490-7499https://doi.org/10.1093/emboj/16.24.7490 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The GAAA tetraloop receptor is an 11-nucleotide RNA sequence that participates in the tertiary folding of a variety of large catalytic RNAs by providing a specific binding site for GAAA tetraloops. Here we report the solution structure of the isolated tetraloop receptor as solved by multidimensional, heteronuclear magnetic resonance spectroscopy. The internal loop of the tetraloop receptor has three adenosines stacked in a cross-strand or zipper-like fashion. This arrangement produces a high degree of base stacking within the asymmetric internal loop without extrahelical bases or kinking the helix. Additional interactions within the internal loop include a U·U mismatch pair and a G·U wobble pair. A comparison with the crystal structure of the receptor RNA bound to its tetraloop shows that a conformational change has to occur upon tetraloop binding, which is in good agreement with previous biochemical data. A model for an alternative binding site within the receptor is proposed based on the NMR structure, phylogenetic data and previous crystallographic structures of tetraloop interactions. Introduction Long-range tertiary interactions are essential for the proper folding and function of large, biologically active RNAs. A highly conserved 11-nucleotide motif containing an internal loop, termed the tetraloop receptor, is known to mediate RNA tertiary folding by providing a binding site for GAAA tetraloops (Costa and Michel, 1995). The GAAA tetraloop receptor domain has been identified in group I and II introns, as well as in the RNase P of some Gram-positive bacteria (Tanner and Cech, 1995). The conservation of the tetraloop receptor domain throughout the evolution of these RNAs suggests an essential role for most of the 11 nucleotides within the motif. In vitro selection experiments using randomized tetraloop receptors revealed that the majority of cloned sequences harbored the canonical motif, with some minor sequence variants that include a C to A substitution within the loop and the exchange of a G·U for an A·C wobble pair (Costa and Michel, 1997). A nearly identical pattern of sequence conservation is found in nature among the group I and II introns and RNase P RNAs that contain the receptor domain (Tanner, 1997). The structure of the tetraloop receptor bound to its cognate GAAA tetraloop, as seen in the crystal structure of the P4–P6 domain of the Tetrahymena group I ribozyme, reveals that most of the conserved nucleotides within the receptor are involved in forming a specific interface with the tetraloop, stabilized by both stacking and hydrogen bonding (Cate et al., 1996a). The three adenosines in the GAAA tetraloop were observed to bind to the tetraloop receptor via a base triplet, a quadruplet, base–sugar and sugar–sugar interactions. Within the receptor, an unusual structural motif was observed, comprised of two consecutive adenines in a co-planar or 'platform' arrangement upon which the GAAA tetraloop stacks. The adenosine platform motif was also observed at sites of intermolecular interactions within the crystal lattice, suggesting that adenosine platforms are a general motif for the mediation of RNA tertiary interactions (Cate et al., 1996b). Most, but not all, of the phylogenetic conservation of the tetraloop receptor sequence can be explained by the interactions observed in the P4–P6 crystal structure (Cate et al., 1996a). For example, the C to A variant in the loop mentioned above is capable of forming an AC platform, a structure that is nearly identical to the AA platform (Zimmermann et al., 1997). The only conserved sequence element which has yet to be structurally rationalized is the C·G pair at the first position (positions 3·21 in our numbering scheme or 222·251 in the Tetrahymena group I intron). Among 35 clones obtained from in vitro selection experiments, 28 had a C·G pair at this position (Costa and Michel, 1997), though no interactions with these bases were found in the P4–P6 crystal structure (Cate et al., 1996a). The basis for macromolecular discrimination between RNA and proteins has been elucidated for a number of systems by comparing the free and bound conformations of the components. In many cases, the interactions between proteins or peptides and RNA result in a conformational rearrangement of the RNA. For example, interactions involving tRNAAspanticodon-synthetase (Ruff et al., 1991), the U1A complex (Allain et al., 1996), the HIV RRE–REV (Battiste et al., 1996; Peterson and Feigon, 1996) and TAT–TAR (Aboul-ela et al., 1995) all involve induced fit or conformational rearrangement upon binding. Similar results are observed for RNA aptamer complexes bound to their ligands (Dieckmann et al., 1996; Jiang et al., 1996; Zimmermann et al., 1997). It remains to be seen whether RNA–RNA tertiary interactions also generally proceed through conformational rearrangements. We have used NMR methods to solve the solution structure of the isolated tetraloop receptor domain, which consists of the phylogenetically conserved 11-nucleotide consensus sequence (Costa and Michel, 1995) embedded within a model 23-nucleotide stem–loop sequence. Instead of the A platform found in the crystal structure of the bound form of the tetraloop receptor (Cate et al., 1996a), the adenines are arranged in a cross-strand stacking arrangement that we call a base zipper. We use molecular modeling to show that an alternative tetraloop-binding site exists within the receptor, based upon the phylogenetic conservation of the tetraloop receptor sequence and previously determined crystallographic structures of GAAA tetraloop interactions (Pley et al., 1994a; Cate et al., 1996a). Results Assignment of proton resonances The sequence of the GAAA tetraloop receptor RNA is shown in Figure 1a. The numbering scheme used in this study is indicated, while the numbering system for the Tetrahymena group I intron is shown in parenthesis (Burke et al., 1987). The two strands were linked by the extra stable UUCG tetraloop, which has been well characterized by NMR (Cheong et al., 1990; Allain and Varani, 1995b). Titrations of up to 25 mM MgCl2 indicated no evidence for magnesium-induced conformational changes; therefore, most spectra were acquired in the presence of 100 mM NaCl. The base-paired imino proton resonances in the stem regions and the UUCG tetraloop (Cheong et al., 1990) are clearly visible, as are the U5, G8 and U17 imino proton resonances (Figure 1b). The exchangeable protons were assigned via sequential NOEs observed in a 2D NOESY spectrum, and were confirmed with 1H-15N HMQC, 1H-15N 2D HMQC-NOESY and 2D HCCCNH TOCSY spectra as previously described (Dieckmann and Feigon, 1997) (data not shown). In the internal loop, a strong imino–imino NOE is observed for G8 and U17 which, in combination with the non-exchangeable NOE data, is consistent with the formation of a G·U wobble pair closing the internal loop. The U5 imino resonance in the internal loop is partially protected from solvent exchange, while the U19 imino resonance is not visible and is therefore exposed to solvent. Figure 1.(a) Sequence and secondary structure of the tetraloop receptor RNA used in this study. The 11 conserved nucleotides for the receptor are in bold. The numbering system is indicated, with the corresponding numbering scheme for the Tetrahymena group I intron tetraloop receptor in parenthesis. (b) 500 MHz 1D 1H NMR spectrum of the imino region of the tetraloop receptor RNA recorded at 274 K. Ninety–six scans of 4096 points were acquired, with a sweep width of 10 000 Hz. The data were zero filled to 8192 points and processed with an exponential filter function with a line broadening of 3.0 Hz. The sample was 1 mM in 450 μl, pH 5.5, in 90% H2O/10% D2O and 100 mM NaCl. Assignments of the imino resonances are indicated. Download figure Download PowerPoint Complete assignments for all of the non-exchangeable proton resonances and their directly bound carbons were obtained using a series of experiments in D2O including homonuclear 2D NOESY, DQF COSY and TOCSY, as well as heteronuclear HCNCH, HCCH TOCSY optimized for adenine H8–H2 correlation, 1H-13C HSQC, 15N long-range HSQC, 3D (1H-13C) HMQC-NOESY and 3D HCCH TOCSY and COSY experiments following previously described protocols (Nikonowicz and Pardi, 1993; Dieckmann and Feigon, 1994, 1997; Pardi, 1995; Varani et al., 1996) (see Materials and methods). Several starting points were available for assigning the two stem regions by their sequential NOE connectivities. Both the UUCG tetraloop and the terminal stem sequence beginning with consecutive G·C base pairs displayed nearly identical chemical shifts to those previously reported (Allain and Varani, 1995a). While the single adenine H2 resonance in the stem (A9) is easily assignable by its sharp, characteristic NOE to the U16 imino proton, the three internal loop adenine H2 protons could only be unambiguously assigned by direct correlation with their corresponding H8 protons, using an HCCH TOCSY experiment optimized for adenine H8–H2 correlation (Legault et al., 1994; Marino et al., 1994). Figure 2 shows a 1H-13C HSQC aligned with the HCCH TOCSY, demonstrating the through-bond correlation between the adenine H2 and H8 resonances. Interestingly, two of the adenine H2 protons in the internal loop (A6 and A18) resonate upfield of the stem A9 H2 proton. These unusually high field chemical shifts may be caused by ring current effects, if the A H2 protons are stacked directly above or below an aromatic ring. Figure 2.(a) Aromatic portion of a 600 MHz 1H-13C HSQC spectrum. A total of 1024 and 512 points were acquired in t2 and t1, respectively, with 16 scans per t1 increment, and a relaxation delay of 1.6 s. The sweep width was 6000 Hz in t2 and 12 000 Hz in t1. The final data matrix was 1024×1024 points and was processed with a 90° shifted squared sine bell filter function. (b) 500 MHz 1H-13C HCCH TOCSY spectrum optimized for AH2–AH8 correlations. 512 and 160 points were acquired in t2 and t1, respectively, with 320 scans per t1 increment, and a relaxation delay of 1.6 s. The sweep width was 5000 Hz in both dimensions. The final data matrix was 1024×1024 points and was processed with a 90° shifted squared sine bell filter function. Lines trace the through-bond correlations between AH2 and AH8 protons. Spectra were recorded at 293 K. The fully 13C,15N-labeled RNA was 1 mM in 200 μl in a Shigemi NMR tube, pH 6.2, 100 mM NaCl in D2O. Download figure Download PowerPoint Sequential NOE connectivities can be traced from the stem regions throughout the internal loop without interruption (Figure 3). The sequential sugar-to-base and base-to-base internucleotide NOEs observed in the 2D NOESY spectrum indicate that there are no extrahelical bases in the internal loop. Analysis of the NOE data as a function of NOESY mixing time suggest that the three spacings between the bases of A6 and A7, U17 and A18, and A18 and U19 are larger than those found in A-form RNA, since the aromatic internucleotide NOEs for these bases are less intense than the ones observed for bases within the Watson–Crick stems. Figure 3.H1′ to aromatic region of a NOESY spectrum (τm = 250 ms) of the tetraloop receptor RNA at 303 K in D2O showing the H1′-base H8/H6 crosspeak region. The base-H1′ sequential connectivities are traced. A total of 1024 and 800 complex points were acquired in t2 and t1, respectively, with 96 scans per t1 increment, and a relaxation delay of 1.6 s. The sweep width was 5000 Hz in both dimensions. The final data matrix was 2048×2048 points and was processed with a Gaussian filter function (line broadening −18 Hz, GB 0.08 in f2 and 0.14 in f1). RNA sample is the same as in Figure 1, except that the pH was raised to 6.2 and the sample transferred into D2O. Download figure Download PowerPoint Torsion angles Analysis of the short mixing time NOESY spectra (50 ms) indicates that all of the bases in the internal loop have glycosidic angles in the anti range, with weak intranucleotide base to H1′ NOEs and strong intranucleotide base to H3′ NOEs (data not shown). The single exception in the molecule is the syn base G14, which is in the UUCG tetraloop (Allain and Varani, 1995b; also data not shown). In addition, the weak or absent H1′–H2′ crosspeaks observed in DQF COSY spectra (JH1,H2 0.2 Angle violations (°) 0 >5 Mean deviation from covalent geometry Bond lengths (Å) 0.01 Angles (°) 2.6 Impropers (°) 0.79 The 20 lowest energy structures are shown in Figure 4a. The conserved 11-nucleotide receptor region is well defined in all 20 low-energy structures and has an r.m.s.d. value for all heavy atoms relative to the mean structure of 1.03 ± 0.28 Å (Table I). At the bottom of the receptor internal loop, a G·U wobble pair forms. The internal loop adenines are arranged in an unusual cross-strand stacking or 'base zipper' motif, with A18 reaching across the internal loop and stacking between A6 and A7, while A6 stacks between A18 and U5 (Figure 4a and b). No hydrogen bonds occur within this zipper region, which appears to be stabilized only by the stacking interactions. The cross-strand stacking interactions in the internal loop are between the six-membered rings of the adenines. The uridines at the top of the internal loop stack between the stem C·G pair and A6, and form a base pair in the majority of the structures (12 out of 20) consisting of a single hydrogen bond between the imino proton of U5 and the O4 oxygen of U19. Figure 4.(a) The 20 lowest energy structures, superimposed upon the heavy atoms of nucleotides 3–8 and 17–21. View is into the minor groove. Non–conserved stem nucleotides and the UUCG tetraloop are purple. The conserved tandem C·G pairs (nucleotides 3–4 and 20–21) are green, U5 and U19 are cyan, A6 and A7 are red, the G8·U17 wobble is yellow and A18 is magenta. (b) Schematic illustration of the tetraloop receptor RNA. Rectangles indicate bases, and stacking interactions are shown as filled black rectangles. Hydrogen bonds are indicated with black lines. Download figure Download PowerPoint The structure correlates very well with NMR data that were not included in the calculations, such as proton exchange rates and chemical shifts. The U5 imino proton is hydrogen-bonded to the O4 carbonyl oxygen of U19 in the majority of the structures, and this imino proton is observed to exchange slowly with the solvent. Conversely, the U19 imino proton points directly out to the solvent, and this imino proton exchanges too rapidly to be observed in 1D NMR spectra. The cross-strand stacking arrangement of the zipper motif provides an explanation for the observed chemical shifts of the AH2 protons. The upfield shifted A6 and A18 H2 protons are stacked directly upon the six-membered rings of A18 and A7, respectively, where they would be subject to ring current effects, while the downfield shifted A7 H2 is exposed to solvent and not stacked. Finally, the chemical shifts observed in 1H-15N HMQC spectra indicate that the adenine amino groups in the internal loop are not hydrogen-bonded, and a 1H-15N HSQC (Sklenář et al., 1994) spectrum suggests that there are no hydrogen-bonded N7 atoms (data not shown). Discussion Comparison of the structures of the free and bound GAAA tetraloop receptor RNA The isolated GAAA tetraloop receptor forms a structure that is quite different from that of the bound tetraloop receptor, which folds into an adenosine platform motif (Cate et al., 1996a). A comparison of the two structures is shown in Figure 5a and b. There are some structural elements common to both structures, including the G·U wobble pair at the bottom and the tandem C·G pairs at the top of the internal loop. Additionally, A7, which corresponds to the 3′ A in the platform motif, stacks upon the G in the G·U wobble pair in both structures, and U5 stacks upon C4 in both structures. Figure 5.Comparison of the structures of the (a) free and (b) bound forms of the tetraloop receptor: (a) is the lowest energy NMR structure of the free receptor; (b) is the crystal structure of the bound tetraloop receptor as observed in the P4–P6 domain of the Tetrahymena group I ribozyme (Cate et al., 1996a). For clarity, the tetraloop is not shown. View is into the major groove. Download figure Download PowerPoint Major differences between the two structures include the location of the three adenines (A6, 7 and 18) and U19. In the free tetraloop receptor, A6 stacks between U5 and A18, while in the bound form of the receptor, A6 would stack upon U17 to form a portion of the adenosine platform. In the solution structure of the free receptor, U19 stacks upon the stem G20, while in the bound form A18 would stack upon G20 and U19 would be extrahelical. The many cross-strand NOEs that we observe in the internal loop precisely define the base zipper and are clearly inconsistent with the formation of an adenosine platform as observed in the P4–P6 crystal structure (Cate et al., 1996a). The solution structure of the free GAAA tetraloop receptor is in good agreement with biochemical data which suggest that a conformational change takes place upon removing the cognate tetraloop (Murphy and Cech, 1994; Cate et al., 1996a). Murphy and Cech demonstrated that mutations in the P4–P6 domain that disrupt the receptor–tetraloop interaction give rise to increased dimethylsulfate (DMS) reactivity at the adenine N1 at position 225 (corresponding to A6 in our numbering scheme), while in the bound form of the receptor this position is protected from DMS modification. Consistent with this data, we find that the A6 N1 position is accessible to solvent in the solution structure of the free receptor, which correlates nicely with the enhanced DMS reactivity at this position when the receptor is probed in its unbound form. Conversely, the A7 N1 (corresponding to the 3′ A226 in the platform), which shows no enhanced reactivity to DMS in the free tetraloop receptor (Murphy and Cech, 1994), is buried in the solution structure. The base zipper is a common structural motif Interstrand stacking interactions are a common structural element in nucleic acids. Several examples of cross-strand stacking interactions exist for both RNA (Szewczak et al., 1993; Wimberly et al., 1993; Pley et al., 1994b; Scott et al., 1995) and DNA (Maskos et al., 1993; Chou et al., 1997). However, the RNA examples are of single cross-strand stacks and not multiple or 'zippered' stacks. Similar base zipper or 'interdigitation' motifs were observed over 20 years ago in tRNAPhe, where interstrand stacking occurs for the six-membered ring of A9 between the six-membered rings of G45 and 46, as well as A21 between G46 and C48, and G18 between G57 and A58 (Robertus et al., 1974). In these base zipper motifs, the 3′ nucleotide on one strand commonly adopts a C2′-endo conformation to allow the accommodation of an interdigited base between sequential nucleotides (Saenger, 1984). We note that spectroscopic data on A18 indicates that it probably is in the S-type conformational range most of the time, which is consistent with the fact that the backbone has to traverse a greater distance at this position to accommodate A7 between U17 and A18. A7 also has a C1′ chemical shift consistent with an S-type sugar pucker, although a range of sugar puckers at this position were obtained from the calculations. Concurrent with this work, another base zipper has been recently identified in the structure of a theophylline-binding RNA aptamer (Zimmermann et al., 1997). Thus, it seems that base zippers are a commonly used RNA structural motif, and it will be interesting to determine how frequently they occur in other RNA internal loop structures. How does a base zipper convert to a platform? The fact that the structure of the free receptor is different from its structure in the context of the tertiary interactions in the P4–P6 crystal structure argues for a conformational rearrangement of the tetraloop receptor upon binding the GAAA tetraloop. Initial folding of the receptor into the base zipper motif appears likely, if one assumes that helical elements fold more quickly than long-range tertiary interactions. In the case of the RNase P tetraloop receptor (Tanner and Cech, 1995), the receptor must fold before the tetraloop, because the receptor portion of the RNA is 5′ to the cognate tetraloop and is transcribed first. The conformational rearrangement required to form the adenosine platform motif is illustrated in Figure 5. The backbone of one strand of the molecule must slide across the major groove to unstack the zipper motif and form the co-planar consecutive adenosines that make up the platform, upon which the tetraloop stacks. During adenosine platform formation, the single hydrogen bond between the U·U mismatch pair must be broken and the 3′U bulged out of the helix. The sequence conservation of the top C3·G21 base pair of the tetraloop receptor has yet to be explained. In the crystal structure, only a single 2′OH from these nucleotides is within hydrogen bonding distance of the bound tetraloop (Cate et al., 1996a). Therefore, the crystal structure does not explain the high degree of conservation of this base pair in nature, which argues for an important function associated with this base pair. Simple stability arguments do not provide a sufficient explanation, since nearest neighbor rules indicate that a C·G pair at this position is less stable than G·C (Serra and Turner, 1995). Interestingly, it has been known for some time that GAAA tetraloops co-vary and interact with tandem C·G pairs (Michel and Westhof, 1990; Jaeger et al., 1994). This interaction has been observed directly in a hammerhead ribozyme crystal structure, in which an interaction between a GAAA tetraloop and tandem C·G pairs was observed within the minor groove of an RNA helix (Pley et al., 1994a). The interaction between the tetraloop and the tandem stem C·G pairs involves a (G·A)·(C·G) base quadruple with the first C·G pair (Pley et al., 1994a). This base quadruple interaction has exactly the same hydrogen bond interactions as the one observed in the P4–P6 crystal structure (Cate et al., 1996a), except that the P4–P6 interactions are with the second C·G pair instead of the first. In other words, the docking of the tetraloops into the tandem C·G pairs differs in register by one C·G base pair between the two crystal structures. Therefore, it is possible that the phylogenetic conservation of the first C·G pair in the tetraloop receptor is to maintain an alternative docking register for the tetraloop. The presence of an alternative docking site for the tetraloop suggests that the interaction with the receptor may not require the formation of an adenosine platform a priori. We propose a model in which the tetraloop initially recognizes the receptor by docking at the first C·G base pair and forming the base quadruplet observed in the hammerhead crystal structure (Pley et al., 1994a). Such an interaction would also be stabilized by a base triple between the second C·G pair and the first A in the GAAA tetraloop as described (Pley et al., 1994a). This could nucleate a structural transition in which the GAAA tetraloop translocates down to the second C·G base pair to form the same type of base quadruplet, and the receptor conformation converts from the A zipper to the A platform. Molecular modeling calculations show that binding of a GAAA tetraloop in the alternative register is sterically feasible. The proposed initial interaction is shown (Figure 6a and c). The model was generated by energy minimization using X-PLOR and the hydrogen bonds described by Pley et al. (1994a) as distance constraints. The position of the tetraloop in the model differs from the one in the crystal structure of Cate et al. (1996a) by one nucleotide in register (Figure 6b and d). Aside from assigning a role for an apparently unrecognized though phylogenetically conserved C·G base pair, the model makes testable predictions. If the alternative binding site is utilized during the course of folding or catalysis, then mutations at the top C·G pair may produce a measurable effect upon one of these steps, even though they would not be expected to interfere with adenosine platform formation. For example, the presence of the top C·G pair may increase the on-rate of the tetraloop for its receptor, if the conformational change is a rate-limiting step for tetraloop binding; or the alternative binding site may be utilized as part of a conformational switch required for a particular step of catalysis. Figure 6.Model for an alternative tetraloop-binding site within the receptor RNA. (a) The model of a GAAA tetraloop docked into the conserved C·G pairs of the receptor, created with a set of distance restraints based on the NMR structure and a previously observed tetraloop interaction (Pley et al., 1994a). View is into the minor groove. (b) The crystal structure of the GAAA·receptor complex within the P4–P6 domain of the Tetrahymena group I ribozyme (Cate et al., 1996a), shown as a comparison. (c) Schematic diagram of the interaction shown in (a). (d) Schematic diagram of the interaction shown in (b). Download figure Download PowerPoint Materials and methods Sample preparation RNA was prepared enzymatically from a DNA template using T7 RNA polymerase (Milligan et al.,