Direct Measurement of Oligonucleotide Substrate Binding To
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Proc. Nati. Acad. Sci. USA Vol. 87, pp. 8187-8191, November 1990 Biochemistry Direct measurement of oligonucleotide substrate binding to wild-type and mutant ribozymes from Tetrahymena (RNA catalysis/gel mobility shift/binding energy/Mg2+ dependence/dissociation constants) ANNA MARIE PYLE, JAMES A. MCSWIGGEN*, AND THOMAS R. CECHt Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215 Contributed by Thomas R. Cech, August 8, 1990 ABSTRACT Like protein enzymes, RNA enzymes (ri- 3' end being determined by a Sca I site in the DNA template). bozymes) provide specific binding sites for their substrates. We This ribozyme is 388 nucleotides in length, and G414 has been now show that equilibrium dissociation constants for complexes deleted in order to study reactions in the presence of exoge- between the Tetrahymena ribozyme and its RNA substrates and nous guanosine. The preparation of this ribozyme and its products can be directly measured by electrophoresis in poly- oligonucleotide substrates has been described (2). Oligonucle- acrylamide gels containing divalent cations. Binding is 103- to otide products were transcribed from the same DNA tem- 104-fold tighter (4-5 kcal/mol at 42C) than expected from plates as the substrates, but ATP was omitted to cause the base-pairing interactions alone, implying that tertiary interac- phage T7 RNA polymerase to terminate before transcription of tions also contribute to energetic stabilization. Binding de- the substrate adenine residues. The carrier oligonucleotide of creases with single base changes in the substrate, substitution unrelated sequence (5'-GCGGUAGGUUGCCCN-3', where of deoxyribose sugars, and lower Mg2+ concentration. Ca2+, N is a heterogeneous terminal nucleotide) and carrier tRNA which enables the ribozyme to fold but is unable to mediate were also prepared by transcription with T7 RNA polymerase efficient RNA cleavage, promotes weaker substrate binding and were gifts from M. Fedor and E. Tinkle (University of than Mg2+. This indicates that Mg2& has special roles in both Colorado, Boulder, CO). substrate binding and catalysis. Mutagenesis of a region near Direct Determination of Binding Constants. Ribozyme di- the internal guide sequence disrupts substrate binding, lutions at 2x final concentration were preincubated at 500C whereas binding is not significantly affected by a mutation of for 10 min before mixing with equal volumes of 1.0 nM the guanosine-binding site. This approach should be generally oligonucleotide substrate (S) or product (P). The oligonucle- useful for analysis of ribozyme variants independent of their otide was 5' end-labeled by treatment with calf intestinal catalytic activities. phosphatase followed by polynucleotide kinase and [y_32p]_ ATP (2). Samples were incubated at the indicated tempera- Ribozymes derived from the self-splicing intervening se- tures for 5 min in 50 mM Tris, pH 7.5/0.1 mM EDTA/10 mM quence (IVS) of Tetrahymena rRNA catalyze phosphodiester NaCI/10 mM MgCI2/3% glycerol/0.05% xylene cyanol and bond substrates (1, 2). The mech- then loaded immediately on a polyacrylamide gel (0.4 mm x cleavage ofoligonucleotide 230 mm x 280 mm) running at 10 W. Binding was unaffected anism for this reaction is similar to the first step in self- by the presence of the dye or glycerol in the mixture (as splicing of the pre-rRNA (3). Substrate recognition is medi- determined by substitution with 5% sucrose). The electro- ated by base-pairing interactions between an internal guide phoresis buffer was 100 mM Tris-Hepes, pH 7.5/0.1 mM sequence (IGS) within the ribozyme and a complementary EDTA/10 mM Mg2+. In addition to electrophoresis buffer, substrate (4-6). A schematic of this interaction is shown in the 10% polyacrylamide gel was prepared with a 30:1 weight Fig. 1. Given this model, one should be able to estimate the ratio of acrylamide to N,N'-methylenebisacrylamide. Gels interaction energy by using rules developed for prediction of were run at constant temperature by clamping to a reservoir duplex stability (7-9). For a circular form of the same IVS, fitted with recirculating coils. Quantitation of free oligonu- however, kinetic results suggest that oligonucleotides may cleotide and its complex was performed on an AMBIS bind to the active site with a greater affinity than expected for radioanalytic scanner. Except where noted, binding of sub- simple duplex formation (10, 11). strate and product oligonucleotides was studied in the ab- Nondenaturing gel electrophoresis has been extensively sence of guanosine, in order to prevent strand scission. used for studying nucleic acid-protein associations (12-17). Additional controls were performed to ensure that the gua- Complexes between RNA moieties have also been observed nosine-independent hydrolysis reaction (21) did not cleave in native gels (18-20). Here we show that ribozyme-substrate significant amounts of the substrate under the conditions of complexes are stable in nondenaturing polyacrylamide gels the binding study. The dissociation constant (Kd) obtained containing Mg2' or Ca2+. The ability to partition free and using different ribozyme preparations varied from 0.7 to 1.3 ribozyme-bound RNA substrate makes it possible to quan- nM for GGCCCUCU. Similar day-to-day variation in Kd was titatively study factors contributing to the energetics of observed using different IVS dilutions of the same ribozyme association. preparation. Studies of oligonucleotide binding in the pres- ence of guanosine were performed at room temperature METHODS under the conditions described above except that the elec- RNA Preparation. The L-21 Sca I ribozyme used in this trophoresis buffer was 50 mM Tris/50 mM borate/0.1 mM study is a shortened form of the Tetrahymena thermophila EDTA, pH 8.3/0.5 mM guanosine/10 mM MgCl2. pre-rRNA IVS, lacking the first 21 and last 5 nucleotides (the Abbreviations: IVS, intervening sequence; IGS, internal guide se- quence. The publication costs of this article were defrayed in part by page charge *Present address: United States Biochemical Corporation, Cleve- payment. This article must therefore be hereby marked "advertisement" land, OH 44122. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 8187 Downloaded by guest on September 30, 2021 8188 Biochemistry: Pyle et al. Proc. Natl. Acad. Sci. USA 87 (1990) G-binding site Kd + GGCCCUCUAAAAA A A A "matched" E-S complex free ribozyme substrate (E) (S) rG '-) GA5 Kd GGq9qyw9t + GGCCCUCU A/ A cA "matched" E- P complex free ribozyme product (E) (P) FIG. 1. Schematic of the interaction between the L-21 Sca I ribozyme and its "matched" substrate and product. In the E-S complex, base-pairingjoins the substrate to a portion ofthe IGS (3'-GGGAGG-5') to form helix P1. After ribozyme-catalyzed cleavage, similar interactions persist in the E'P complex. The guanosine-binding site is depicted above the P1 helix, where it is poised to effect scission of the substrate U-A bond. Two of the three italicized adenine residues are deleted in the J1/2-2A variant of the ribozyme. Determination of Binding by Competition. The association tration ofsubstrate (0.05-1 nM), the volume ofsample loaded of GGCCCGCU, GGCCCGCUA5, and d(GGCCCUCU) to on the gel, or the electrophoresis time. Carrier RNA mole- the ribozyme was studied by competition with the radiola- cules, including tRNA and an unstructured oligonucleotide of beled probe 32P-GGCCCUCU. The validity of the competi- unrelated sequence (both at 400 nM in nucleotides), did not tion assay was verified in two ways. Unlabeled GGCCCUCU change the Kd ofCCCUCU and therefore did not compete for was found to compete with 32P-GGCCCUCU in binding to the binding site. This confirms that complementarity between the ribozyme at 250C. A calculated Kd for the inhibitor was the active site and oligonucleotides is essential for associa- found to be 1.5 nM. Second, the binding of 32P-GGCCCGCU tion. was studied directly at several temperatures and found to be Equilibrium Dissociation Constants of Oligonucleotide- only a factor of 3 weaker than that determined by competi- Ribozyme Complexes. The dissociation constant of GGC- tion. The competitive Kd determination is more reliable due CCUCU obtained by inspection of the gel midpoint (Fig. 2A) to smearing of the relatively unstable 32P-GGCCCGCU- or the fit to a theoretical binding curve (Fig. 2B) is =1 nM at ribozyme complex in the gel, which artifically lowers the 250C and 42TC. This value corresponds to a binding free observed Kd. energy of -12.2 (250C) or -12.9 (420C) kcal/mol, which is Free Energy Calculations. The observed free energies were =0.4 kcal/mol greater than the energy calculated by consid- determined as AG' = -RT ln(l/Kd), where R = 0.00198 ering base-pairing alone at 250C and 4.0 kcal/mol greater at kcal-mol-1K-1 and T is in kelvins. The calculated free 420C. energies were determined by AGO = AHM - TAS0 using AH0 The structure of RNA substrates and products was varied and ASO values from refs. 8 and 9. The terminal A-G in order to learn more about specific binding requirements. A mismatch and the terminal G-U pair were included in the single base substitution at a position three nucleotides from calculation. the 3' end of the product adversely affects binding to the ribozyme. This is shown by comparison of 1 with 4 and 2 with 5 in Table 1. However, the binding remains tighter than that RESULTS calculated for a simple mismatched helix, 4.5 kcal/mol of Method for Direct Determination of Binding Constants. The extra binding energy at 250C, and 5.5 kcal/mol at 420C for 4. oligonucleotide GGCCCUCU, which forms a "matched" There is little difference in the Kd of reaction substrates and duplex with the guide sequence of the ribozyme, represents their corresponding products.