Portal de Periódicos da CAPES

The global structure of the VS ribozyme

2002; Springer Nature; Volume: 21; Issue: 10 Linguagem: Inglês

10.1093/emboj/21.10.2461

1460-2075

Daniel A. Lafontaine, D. Norman, David M.J. Lilley,

RNA modifications and cancer

Article15 May 2002free access The global structure of the VS ribozyme Daniel A. Lafontaine Daniel A. Lafontaine Cancer Research UK Nucleic Acid Structure Research Group, Department of Biochemistry, MSI/WTB Complex, The University of Dundee, Dundee, DD1 5EH UK Search for more papers by this author David G. Norman David G. Norman Cancer Research UK Nucleic Acid Structure Research Group, Department of Biochemistry, MSI/WTB Complex, The University of Dundee, Dundee, DD1 5EH UK Search for more papers by this author David M.J. Lilley Corresponding Author David M.J. Lilley Cancer Research UK Nucleic Acid Structure Research Group, Department of Biochemistry, MSI/WTB Complex, The University of Dundee, Dundee, DD1 5EH UK Search for more papers by this author Daniel A. Lafontaine Daniel A. Lafontaine Cancer Research UK Nucleic Acid Structure Research Group, Department of Biochemistry, MSI/WTB Complex, The University of Dundee, Dundee, DD1 5EH UK Search for more papers by this author David G. Norman David G. Norman Cancer Research UK Nucleic Acid Structure Research Group, Department of Biochemistry, MSI/WTB Complex, The University of Dundee, Dundee, DD1 5EH UK Search for more papers by this author David M.J. Lilley Corresponding Author David M.J. Lilley Cancer Research UK Nucleic Acid Structure Research Group, Department of Biochemistry, MSI/WTB Complex, The University of Dundee, Dundee, DD1 5EH UK Search for more papers by this author Author Information Daniel A. Lafontaine1, David G. Norman1 and David M.J. Lilley 1 1Cancer Research UK Nucleic Acid Structure Research Group, Department of Biochemistry, MSI/WTB Complex, The University of Dundee, Dundee, DD1 5EH UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:2461-2471https://doi.org/10.1093/emboj/21.10.2461 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The VS ribozyme comprises five helical segments (II–VI) in a formal H shape, organized by two three-way junctions. It interacts with its stem–loop substrate (I) by tertiary interactions. We have determined the global shape of the 3–4–5 junction (relating helices III–V) by electrophoresis and FRET. Estimation of the dihedral angle between helices II and V electrophoretically has allowed us to build a model for the global structure of the complete ribozyme. We propose that the substrate is docked into a cleft between helices II and VI, with its loop making a tertiary interaction with that of helix V. This is consistent with the dependence of activity on the length of helix III. The scissile phosphate is well placed to interact with the probable active site of the ribozyme, the loop containing A730. Introduction RNA catalysis is important for cellular function. A number of RNA processing events, including tRNA maturation (Guerrier-Takada et al., 1983), are catalysed by ribozymes. In addition, protein synthesis appears to be catalysed by the 23S rRNA of the large ribosomal subunit (Nissen et al., 2000), and it is very likely that mRNA splicing will turn out to be RNA catalysed (Gordon et al., 2000; Yean et al., 2000). However, the fundamental chemical mechanisms of these processes are still poorly understood. The small nucleolytic ribozymes have been valuable in the analysis of basic mechanisms of RNA catalysis, owing to their small size and simplicity. These fall into four groups, the hammerhead (Epstein and Gall, 1987; Forster and Symons, 1987), hairpin (Buzayan et al., 1986), hepatitis delta virus (HDV) (Sharmeen et al., 1988) and Varkud satellite (VS) (Saville and Collins, 1990) ribozymes. While each has a distinct structure, they all carry out essentially the same reaction, i.e. the cleavage of a specific phosphodiester linkage by a transesterification reaction involving attack of the neighbouring 2′-oxygen with departure of the 5′-oxygen. The reaction is accelerated by a factor of 105–106 by the ribozymes (Hertel et al., 1997). Where analysed, it has been found that the chirality of the phosphorus becomes inverted in the course of the reaction (van Tol et al., 1990; Koizumi and Ohtsuka, 1991), suggesting an SN2 mechanism. The reaction might be subject to a number of different catalytic strategies, including deprotonation of the 2′-hydroxyl group, facilitation of the trajectory into the in-line transition state, charge stabilization in the transition state and stabilization and/or protonation of the oxyanion leaving group. Nucleobases could act in general acid–base catalysis, exemplified by a cytosine in the HDV ribozyme (Perrotta et al., 1999; Nakano et al., 2000), while metal ions could act in general acid–base and/or electrophilic catalysis. Any such process, however, requires the correct folding of the RNA and association with the substrate to create the correct local environment in which the catalysis can proceed. The VS ribozyme is the largest of the known nucleolytic ribozymes. It is found in the RNA transcribed from the Varkud satellite DNA of Neurospora mitochondria (Kennell et al., 1995). Cleavage occurs within the substrate stem–loop (stem I) which can be unlinked from the remaining ribozyme that consists of five helical sections (II–VI) (Figure 1). In the trans-acting form of the ribozyme, the association with the substrate occurs largely through tertiary interactions, including a long-range loop–loop interaction between helices I and V (Rastogi et al., 1996). It has also been suggested that the secondary structure of the substrate is altered upon binding to the ribozyme (Andersen and Collins, 2000). In addition to the cleavage reaction, the ribozyme can also catalyse the reverse, ligation reaction (Saville and Collins, 1991). Figure 1.The VS ribozyme. (A) The sequence and deduced secondary structure of the VS ribozyme (Beattie et al., 1995), drawn in its cis-acting form. The position of cleavage is indicated by the arrow. The broken line indicates a proposed tertiary interaction between the loops of helices I and V (Rastogi et al., 1996). (B) Conversion into a trans-acting ribozyme and substrate. Download figure Download PowerPoint Crystal structures have been obtained for the hammerhead (Pley et al., 1994; Scott et al., 1995), hairpin (Rupert and Ferré-D‘Amaré, 2001) and HDV (Ferré-D’Amaré et al., 1998) ribozymes, and these have been extremely useful in refining ideas concerning catalytic mechanisms. However, there is at present relatively little structural information on the VS ribozyme. The secondary structure of the ribozyme section (Beattie et al., 1995) shows that the five helical sections are organized by two three-way junctions as a formally H-shaped structure (Figure 1). Experience with a number of RNA species indicates that helical junctions can play an important role as structural features that organize the conformation on a large scale. For example, the hairpin ribozyme requires the association of two loops for activity; these are carried on two adjacent arms of a perfect four-way junction, the presence of which greatly enhances the efficiency of folding into the active form of the ribozyme (Murchie et al., 1998; Walter et al., 1999; Zhao et al., 2000). We have therefore embarked on an examination of the structure of the two three-way junctions of the VS ribozyme, with the goal of defining the global fold of the complete five-helix ribozyme. In a previous paper, we presented the structure of the lower (2–3–6) junction (Lafontaine et al., 2001a) based on comparative gel electrophoresis, long-range distance information derived from FRET, and homology-based modelling. We found that this junction undergoes metal ion-induced folding into a structure in which helices III and VI are co-axially stacked, and a small angle is subtended between helices II and VI. In the present work, we have determined the global shape of the 3–4–5 junction of the ribozyme when folded in Mg2+ ions, and the dihedral angle between the two junctions. This defines the general fold of the ribozyme, and allows us to deduce the location of the substrate stem–loop. We propose that the substrate stem–loop is docked into the cleft between helices II and VI, where it interacts with the probable active site of the ribozyme, the A730 loop (Lafontaine et al., 2001b). Results and discussion The global structure of the 3–4–5 junction The overall shape of the VS ribozyme, comprising helices II–VI, will be largely determined by the conformation of the two three-way helical junctions. We have previously determined the global shape of the 2–3–6 junction, and we have therefore now turned to the remaining 3–4–5 junction that relates helices III, IV and V. This is an HS1HS5HS2 junction (Lilley et al., 1995), i.e. it contains one or more formally unpaired nucleotides linking each of the pairs of helices around the centre. Each of the helices comprises three or more uninterrupted Watson–Crick base pairs from the centre of the junction. We have analysed the trajectories of the arms around the junction using comparative gel electrophoresis and FRET (Lilley, 2000). Analysis of the structure of a three-way junction by comparative gel electrophoresis requires the derivation of the three species comprising two long (extended by randomly chosen sequences to a total length of 40 bp) and one short (11 bp) helical arm, and comparison of their electrophoretic mobilities in a polyacrylamide gel under non-denaturing conditions. The relative mobilities can be related directly to the size of the angle subtended between the two long arms. The component oligonucleotides were generated by transcription, and thus the molecules were entirely composed of RNA. All three helices were perfectly base paired, and thus the bulged adenine of helix III was removed. These are named from the shortened arm; e.g. ΔIII has long arms IV and V, and a short arm III. Electrophoresis was performed in the presence of 0 and 3 mM Mg2+ ions (Figure 2A and B). It can be seen that, in common with most such branched nucleic acids, the structure changes on addition of divalent metal ions. In the absence of added ions, the mobilities increase in the order ΔIII<ΔV<ΔIV, indicating that the angles between helices increase in magnitude in the order IV–V<III–IV<III–V. This corresponds to the size of the unpaired stretch of nucleotides at the three points, and is therefore consistent with an extended structure with no interhelical stacking of arms. This is typical for three-way junctions in both RNA (Bassi et al., 1995, 1997; Lafontaine et al., 2001a) and DNA (Welch et al., 1993). Upon addition of Mg2+ ions, the pattern of mobilities is consistent with a global structure in which the largest angle is subtended between helices III and IV, and the smallest angle lies between helices III and V. Figure 2.The global structure of the 3–4–5 junction. (A and B) Comparative gel electrophoresis. The three possible species having two long arms of 40 bp and one shorter arm of 11 bp were created from transcribed RNA, and named by the shortened arm (e.g. species ΔIII has arm III of 11 bp). The three species were electrophoresed in a polyacrylamide gel in the presence of the indicated metal ion concentrations. The junctions were radioactively 5′-32P-labelled, and the different species were revealed by phosphorimaging of dried gels. The experiment was repeated in the presence of 0 (A) and 3 mM (B) Mg2+ ions. The differences in the patterns of mobility clearly indicate an ion-induced structural transition that alters the global structure of the junction. The interpretations of the mobility patterns are shown to the right of the phosphorimages, corresponding to the global structures illustrated in (E) (see text). (C and D) FRET analysis. We have constructed three species with three arms of 11 bp, each carrying donor (fluorescein) and acceptor (Cy3) fluorophores attached to the 5′ termini of two helical arms. The species are named according to the helical arms carrying the donor and acceptor, in that order. Histograms of FRET efficiency (EFRET) for three end-to-end vectors, measured in the presence of 2 μM (C) or 3 mM (D) Mg2+ ions, are shown. Relative FRET efficiencies agree qualitatively with the global model illustrated in (E). (E) A model for the global folding of the VS 3–4–5 junction. The structure is extended at low divalent ion concentration. On addition of 3 mM Mg2+ ions, the structure undergoes a folding transition that involves movement of helix V, shortening the III–V end-to-end distance. Download figure Download PowerPoint The structure of the 3–4–5 junction was also analysed using FRET. Efficiency of energy transfer (EFRET) between donor–acceptor pairs separated by distance R is given by (Förster, 1948): In this approach, we have compared the efficiency of energy transfer between fluorescein and Cy3 fluorophores (for which R0 = 56 Å; Norman et al., 2000) attached to the 5′ termini of different pairs of helical arms, each 11 bp in length (omitting A718). FRET efficiencies were measured in the presence of 2 μM and 3 mM Mg2+ ions, and the results are presented in Figure 2C and D. The differences between the two patterns of efficiencies confirm the dependence of the structure on divalent metal ions. Moreover, if a simple relationship between end-to-end distance and FRET efficiency is assumed, the results are in excellent agreement with those of the comparative gel electrophoresis. Thus, both methods indicate that the junction adopts an extended structure in the absence of divalent metal ions, and undergoes a folding process on addition of Mg2+ ions. The large angle subtended between helices III and IV might indicate co-axial stacking of these arms; the value of EFRET = 0.18 for the III–IV vector is similar to that measured for a 22 bp duplex (Lafontaine et al., 2001a), where EFRET = 0.20. Ion-induced folding of the 3–4–5 junction The folding of junction 3–4–5 may be followed via the change in EFRET for chosen end-to-end vectors on titration of Mg2+ ions (Figure 3). We have compared vectors III–V and IV–V, since these become shorter and longer, respectively, on folding. Similar analysis of the III–IV vector shows a small change in EFRET at a lower metal ion concentration (a reduction of 0.01 occurring at 0–10 nM), corresponding to a different aspect of the transition (data not shown). The data for vectors III–V and IV–V have been fitted to a two-state conformational transition induced by the binding of Mg2+ ions with a Hill coefficient n and an apparent association constant KA. From these, we can calculate the Mg2+ ion concentration ([Mg2+]1/2 = (1/KA)1/n) at which the transition is 50% complete. The proportion of folded junction (α) is given by: Figure 3.Folding of the VS junction 3–4–5 as a function of Mg2+ concentration. (A) Scheme of the proposed folding of the junction. In this experiment, we analyse the shortening of the III–V vector (filled circle) and the concomitant lengthening of the IV–V vector (open circle). (B) FRET efficiencies for the III–V (filled circles) and IV–V (open circles) vectors are plotted as a function of Mg2+ concentration. The experimental data were fitted (lines) by regression to a two-state model where binding of metal ions induces a structural change. Download figure Download PowerPoint Good fits were obtained with this simple model, giving a value of n = 1.0 ± 0.1 and 0.9 ± 0.1, and [Mg2+]1/2 = 260 and 320 μM, for the vectors 3–5 and 4–5, respectively. Thus the 3–4–5 junction folds in response to the non-cooperative binding of Mg2+ ions. We found previously that the folding of the 2–3–6 junction is also induced by non-cooperative binding of Mg2+ ions (Lafontaine et al., 2001a). The [Mg2+]1/2 was a little lower for the 2–3–6 junction at ∼100 μM, but in a similar range nevertheless. Thus the folding properties of the two three-way junctions of the VS ribozyme are similar, and the complete ribozyme should therefore fold over a relatively narrow ionic concentration range. Sequence changes in the 3–4–5 junction affect catalytic activity We have made substitutions in all the nucleotides that form the 3–4–5 junction, and examined the effect on the cleavage rate in the context of the complete trans-acting ribozyme (Figure 4). Cleavage rates were measured under single-turnover conditions for the ribozyme 1 plus substrate 1 combination (Lafontaine et al., 2001a), and the observed rate constants are collected in Table I. Similar studies have been carried out recently on the cis-acting form of the ribozyme (Sood and Collins, 2001). Figure 4.Effect of sequence changes at the 3–4–5 helical junction on ribozyme cleavage activity in trans. (A) The sequence of the 3–4–5 junction. (B) Separation of substrate and product for the natural ribozyme and selected sequence variants. Radioactively 5′-32P-labelled substrate (∼1 nM) was incubated with an excess of ribozyme (1 μM) for 15 min in the presence of 10 mM Mg2+ ions. At this ribozyme concentration, the observed cleavage rates include contributions from both complex formation and catalysis. After termination, the substrate (arrowed, sub) and product (prod) were separated by gel electrophoresis and visualized by autoradiography. Tracks 1–3, control experiments; 1, unincubated substrate; 2, substrate incubated alone with Mg2+ ions; 3, substrate plus ribozyme incubated in the absence of Mg2+ ions; tracks 4–8, substrate plus ribozyme incubated in the presence of Mg2+ ions; 4, natural sequence; 5, U664A; 6, ΔU686; 7, U710C; 8, A712U. (C) Progress curves for cleavage reactions. Plots of cleaved fraction as a function of time for natural sequence (filled circles) and U664A (open squares); ΔU686 (filled squares); U710C (filled triangles); A712U (open circles) ribozymes. The time scale has been chosen to show the progress of the variant ribozymes, and thus the data for the natural ribozyme are compressed. (D) The effect of deletion of U686 on the conformation of the 3–4–5 junction. Comparative gel electrophoresis was carried out on this variant analogously to that in Figure 2. The pattern of mobilities is interpreted in terms of the global structure shown schematically on the right. Download figure Download PowerPoint Table 1. Effects of sequence changes in the 3–4–5 junction and surrounding helices on cleavage activity VS ribozyme variant kobs/min Natural sequence 1.0 Mutations in the 3–4–5 junction U664A 0.12 C665A 0.015 U686A 0.006 ΔU686 <0.001 U710C 0.03 U710A 0.002 G711U 0.9 A712U 0.005 A712G 0.006 U713A 0.02 U714A 0.14 Proximal base pairs C663G:G715C 0.27 C666G:G685C 0.03 C687G:G709C 0.008 Stem III U659A:A720U 0.8 C660G:G719C 1.2 A661U:U717A 0.6 C662G:G716C 0.6 Stem III reversea 0.04 Stem III reverse, except A718b 0.5 A718 reversec 0.12 ΔA718d 0.14 +A after C660e 0.77 Length 5 bpf <0.001 Length 6 bpf 0.14 Length 7 bpf 0.85 Length 8 bpf 0.09 Length 9 bpf 0.0037 Length 10 bpf <0.001 Stem IV Stem IV, 6 bpg 0.49 Stem IV, 4 bph 0.61 Stem IV, 2 bpi 0.012 Stem V U705Cj 0.9 U708Cj 0.9 Stem V, 3 bpk <0.001 Stem V, 5 bpl <0.001 Stem V, 7 bpm 0.047 Stem V, 10 bpn 0.031 Stem V, 11 bpo 0.0032 Stem V, 12 bpp <0.001 Stem V, 13 bpq 2. Even reversal of all 6 bp, while retaining the adenine bulge (A718) on the 3′ strand only led to a 2-fold reduction in activity. However, when the entire sequence of helix III was reversed including the adenine bulge (thus transferring the bulge to the 5′ strand), this led to a 25-fold reduction in cleavage rate. In contrast, complementation of the adenine bulge (making a perfect 7 bp helix) had almost no effect on activity. Thus the sequence of stem III is completely unimportant for ribozyme activity; the only feature of significance is the orientation of the bulge when present. However, the length of this helix is very important, as we discuss below. Global structure of the VS ribozyme (helices II–V) The basic structure of the ribozyme may be constructed by bringing together the two three-way junctions through their common helix III, generating an approximately co-linear axis for helices IV, III and VI (modelled below; refer to Figure 6). We have established the global structures of both junctions, but one main uncertainty remains. The rotational directions of helices II and V with respect to helix III are not known for the junctions individually, and thus the dihedral angle between these two arms in the five-helix ribozyme is unknown. We have therefore devised an electrophoretic experiment designed to estimate this angle in the complete ribozyme. The principle of this experiment was to compare a series of species in which the longest arms were stems II and V. By varying the length of stem III, we could systematically change the dihedral angle between helices II and V. This would alter the global shape of the molecule, and thus the expected electrophoretic mobility. Figure 5.Estimation of the dihedral angle between stems II and V using gel electrophoresis. (A) This analysis employed a series of variant ribozymes comprising elongated helices II and V, and short helices IV and VI, and a perfectly base paired helix III that varied in length from 6 to 20 bp, constructed from transcribed RNA. Changing the length of the central helix III would have the effect of altering the angle between the two three-way junctions, and thus the dihedral angle between helices II and V. It would be expected that electrophoretic mobility would depend on this angle. (B) Relative electrophoretic mobility of the species in polyacrylamide. Autoradiograph of the radioactive species as a function of the length of helix III. Note the sinusoidal modulation of mobility. (C) Plot of electrophoretic mobility as a function of the length of helix III. The points are the experimentally measured mobilities, with the error bars indicating the uncertainty in estimating the centres of the bands. These data have been fitted to Equation 3 using a simple geometric model from which we can calculate the end-to-end distance for each length of stem III. The dihedral angle for any chosen stem III length can be calculated from this analysis. Download figure Download PowerPoint Using RNA molecules transcribed from DNA templates constructed by PCR, we have g