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Secondary Structure Mapping of an RNA Ligand That Has High Affinity for the MetJ Repressor Protein and Interference Modification Analysis of the Protein-RNA Complex

1999; Elsevier BV; Volume: 274; Issue: 4 Linguagem: Inglês

10.1074/jbc.274.4.2255

1083-351X

Alistair McGregor, James B. Murray, C.J. Adams, Peter G. Stockley, Bernard A. Connolly,

Advanced biosensing and bioanalysis techniques

The secondary structure of an RNA aptamer, which has a high affinity for the Escherichia coli MetJ repressor protein, has been mapped using ribonucleases and with diethyl pyrocarbonate. The RNA ligand is composed of a stem-loop with a highly structured internal loop. Interference modification showed that the bases within the internal loop, and those directly adjacent to it, are important in the binding of the RNA ligand to MetJ. Most of the terminal stem-loop could be removed with little effect on the binding. Ethylation interference suggests that none of the phosphate groups are absolutely essential for tight binding. The data suggest that the MetJ binding site on the aptamer is distinct from that of the natural DNA target, the 8-base pair Met box. The secondary structure of an RNA aptamer, which has a high affinity for the Escherichia coli MetJ repressor protein, has been mapped using ribonucleases and with diethyl pyrocarbonate. The RNA ligand is composed of a stem-loop with a highly structured internal loop. Interference modification showed that the bases within the internal loop, and those directly adjacent to it, are important in the binding of the RNA ligand to MetJ. Most of the terminal stem-loop could be removed with little effect on the binding. Ethylation interference suggests that none of the phosphate groups are absolutely essential for tight binding. The data suggest that the MetJ binding site on the aptamer is distinct from that of the natural DNA target, the 8-base pair Met box. S-adenosylmethionine human immunodeficiency virus systematic evolution of ligands by exponential enrichment diethyl pyrocarbonate. Several genes involved in the biosynthesis of methionine inEscherichia coli are transcriptionally regulated by the methionine repressor, MetJ (1Saint-Girons I. Parsot C. Zakin M.M. Barzu O. Cohen G.N. CRC Crit. Rev. Biochem. 1988; 23: S1-S42Crossref PubMed Scopus (86) Google Scholar). The basic interaction occurs between a homodimer of the 12-kDa MetJ repressor subunits and an 8-base pair sequence that constitutes a Met box (2Saint-Girons I. Duchange N. Cohen G.N. Zakin M.M. J. Biol. Chem. 1984; 259: 14282-14285Abstract Full Text PDF PubMed Google Scholar). The Met box is a tandem repeat that occurs between two and five times in natural operators and to which additional repressor dimers bind in a cooperative manner (3Phillips S.E.V. Manfield I. Parsons I.D. Davidson B.E. Rafferty J.B. Somers W.S. Margarita D. Cohen G.N. Saint-Girons I. Stockley P.G. Nature. 1989; 341: 711-715Crossref PubMed Scopus (96) Google Scholar). The protein has a βαα topology where the β-strands from each subunit intertwine to form an anti-parallel β-ribbon, which lies in the major groove of the target DNA. Binding specificity is largely determined by hydrogen bonds between the side chains of lysine 23 and threonine 25 of the β-strands and 4 purine bases in each Met box. Many hydrogen bonds are also made between the protein and the DNA phosphate backbone, and there is evidence that the conformation of the backbone is also important for binding (4Somers W.S. Phillips S.E.V. Nature. 1992; 359: 387-393Crossref PubMed Scopus (284) Google Scholar). Binding of the MetJ repressor to DNA is modulated by S-adenosylmethionine (AdoMet),1 which markedly increases the affinity of MetJ for its target sequence. AdoMet and the DNA are bound on opposite faces of the protein, and binding of AdoMet does not significantly perturb the protein structure. The increased affinity of the holorepressor appears to be due to a long range electrostatic effect caused by the positively charged tertiary sulfur atom of AdoMet (5Phillips K. Phillips S.E.V. Structure. 1994; 2: 309-316Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 6Parsons I.D. Persson B. Mekhalfia A. Blackburn G.M. Stockley P.G. Nucleic Acids Res. 1995; 23: 211-216Crossref PubMed Scopus (60) Google Scholar). Over the last decade in vitro selection and amplification techniques have been developed to isolate tight binding nucleic acid ligands for a wide range of target molecules (7Tuerk C. Gold L. Science. 1990; 249: 505-510Crossref PubMed Scopus (8173) Google Scholar, 8Ellington A.D. Szostak J.W. Nature. 1990; 346: 818-822Crossref PubMed Scopus (7739) Google Scholar). RNA molecules in particular have been isolated for a diverse set of targets, including proteins (9Schneider D. Tuerk C. Gold L. J. Mol. Biol. 1992; 228: 862-869Crossref PubMed Scopus (115) Google Scholar), amino acids (10Connell G.J. Illangeskare M. Yarus M. Biochemistry. 1993; 32: 5497-5502Crossref PubMed Scopus (168) Google Scholar), and nucleotides (11Sassanfer M. Szostak J.M. Nature. 1993; 364: 550-553Crossref PubMed Scopus (514) Google Scholar). These RNA aptamers potentially have very widespread applications as lead compounds in therapeutic situations, e.g. RNA-based inhibitors of the type I human immunodeficiency virus (HIV-I) reverse transcriptase (12Tuerk C. MacGougal S. Gold L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6988-6992Crossref PubMed Scopus (372) Google Scholar, 13Burke D.H. Scates L. Andrews K. Gold L. J. Mol. Biol. 1996; 264: 650-666Crossref PubMed Scopus (97) Google Scholar) or as ligands in diagnostic kits and biosensors. Despite their importance we have relatively little structural information on the ways in which aptamers interact with protein targets. Recently, one of our laboratories reported the first x-ray crystal structure for an aptamer bound to the RNA bacteriophage MS2 coat protein (14Convery M.A. Roswell S. Stonehouse N.J. Ellington A.D. Hirao I. Murray J.B. Peabody D.S. Phillips S.E.V. Stockley P.G. Nat. Struct. Biol. 1998; 5: 133-139Crossref PubMed Scopus (118) Google Scholar). This structure revealed that the aptamer is bound to the natural RNA binding site of the protein, and although being based on differing primary and secondary structures, the aptamer is able to mimic most of the key recognition contacts of the natural ligand. To extend our knowledge of the way in which aptamers interact with their protein targets, we have isolated tight binding RNA ligands for the E. coli MetJ repressor, a sequence-specific DNA-binding protein with no known RNA-binding function.2 Previously, we have shown that systematic evolution of ligands by exponential enrichment (SELEX) with degenerate DNA ligands results in isolation of tight binding DNA molecules based on the Met box consensus (16He Y.-Y. Stockley P.G. Gold L. J. Mol. Biol. 1996; 255: 55-66Crossref PubMed Scopus (38) Google Scholar), a situation analogous to the isolation of RNA aptamers for the MS2 coat protein (9Schneider D. Tuerk C. Gold L. J. Mol. Biol. 1992; 228: 862-869Crossref PubMed Scopus (115) Google Scholar). However, a high proportion of the SELEX-RNA ligands isolated by binding to MetJ contained the consensus sequence 5′-UGCAUACUCGUUN(3–16)A(G)GCAUUGCAGCA-3′, 2Y.-Y. He, A. L. Ellison, I. D. Parsons, P. G. Stockley, and L. Gold, manuscript in preparation. which is unrelated to the Met box DNA target. Remarkably, several of these ligands bind more tightly to an apo-MetJ dimer than does the DNA consensus, a two Met box site interacting with two cooperatively bound holorepressors (3Phillips S.E.V. Manfield I. Parsons I.D. Davidson B.E. Rafferty J.B. Somers W.S. Margarita D. Cohen G.N. Saint-Girons I. Stockley P.G. Nature. 1989; 341: 711-715Crossref PubMed Scopus (96) Google Scholar). Therefore, it is of interest to determine how such RNA aptamers interact with MetJ. Do they mimic the binding of the natural DNA Met box (as for the MS2 coat protein with RNA ligands and MetJ with DNA ligands), or alternatively, do they represent novel solutions to the MetJ recognition problem? Inhibition of DNA binding proteins by RNA aptamers is likely to be of very general interest. DNA-binding proteins constitute a large group of potential drug targets, and RNA SELEX offers a route to generate tight binding inhibitors of such molecules. Here a number of techniques have been employed both to determine the solution structure of the highest affinity RNA ligand and to probe the nature of the interaction between the two macromolecules. In particular we have attempted to determine the regions of the RNA aptamer that are involved in binding to the protein. Similar approaches have been used to good effect to study the inhibition of HIV-I reverse transcriptase with RNA ligands (17Green L. Waugh S. Binkley J.P. Hostomska Z. Hostomsky Z. Tuerk C. J. Mol. Biol. 1995; 247: 60-68Crossref PubMed Scopus (48) Google Scholar). In the absence of high resolution structural data for the RNA-protein complex, these studies provide a useful insight into how the aptamer complex differs from the DNA operator complex with the same protein. RNA was synthesized on an Applied Biosystems 391 synthesizer usingt-butyldimethylsilyl RNA-phosphoramidites (ChemGenes, Stoke on Trent, UK). Ancillary RNA synthesis reagents were supplied by Applied Biosystems (Warrington, UK). The standard 1.0-μmol DNA synthesis cycle was used with a longer coupling and capping periods of 360 s and 40 s, respectively. Syntheses were carried out trityl-off. Protecting groups, other than the silyl group, were removed by treatment with 2 ml of fresh methanolic ammonia at 37 °C for 20 h. Desilylation was performed by dissolving the RNA in 500 μl of dimethyl sulfoxide (Aldrich) and treating the solution with 500 μl of triethylamine trihydrofluoride (Aldrich) at room temperature for 48 h. The RNA was desalted on a NAP-25 column (Amersham Pharmacia Biotech) prior to ion-exchange high performance liquid chromatography using a NucleoPac PA-100 column (4 × 250 mm) (Dionex, Camberley, UK). The column was developed, at 55 °C, with an ammonium acetate gradient (0–1.5 m, pH 7.5) in solutions containing 10% (v/v) acetonitrile. The required fraction was dried in a rotary evaporator resuspended in sterile water and desalted as above. RNAs containing a hexaethylene glycol spacer were prepared as above using 18-O-dimethoxytritylhexaethylene glycol, 1-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite (Glen Research, Sterling, VA). Oligoribonucleotides containing 4-thiouridine were synthesized as above with one additional step. Immediately following synthesis the support-bound oligomers were treated with 1 ml of 0.3 m1,8-diazabicyclo[5.4.0]undec-7-ene (Aldrich) in dry acetonitrile for 3 h at room temperature to remove the cyanoethyl group that protects the thiol (18Nikiforov T.T. Connolly B.A. Tetrahedron Lett. 1992; 33: 2379-2382Crossref Scopus (31) Google Scholar). The 1,8-diazabicyclo[5.4.0]undec-7-ene was then carefully decanted, and the support was extensively rinsed with acetonitrile, prior to treatment with methanolic ammonia. The 4-thiouridine phosphoramidite was synthesized as reported previously (19Adams C.J. Murray J.B. Arnold R.J.P. Stockley P.G. Tetrahedron Lett. 1994; 35: 765-768Crossref Scopus (27) Google Scholar). All oligoribonucleotides were stored at −20 °C. Base composition analysis was carried out as described previously (20Eadie J.S. McBride L.J. Efcavitch J.W. Hoff L.B. Cathcart R. Anal. Biochem. 1987; 165: 442-447Crossref PubMed Scopus (94) Google Scholar), and the results were consistent with the predicted values for the oligoribonucleotide. Oligoribonucleotides were labeled, at their 5′-ends using [γ-32P]ATP (3000 Ci/mmol; Amersham) and T4 polynucleotide kinase (Amersham Pharmacia Biotech) as described previously (19Adams C.J. Murray J.B. Arnold R.J.P. Stockley P.G. Tetrahedron Lett. 1994; 35: 765-768Crossref Scopus (27) Google Scholar). Alternatively, the 3′-ends were labeled by the ligation of [5′-32P]pCp (3000 Ci/mmol; Amersham Pharmacia Biotech) to the 3′-end using RNA ligase (Amersham Pharmacia Biotech) (21Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 5.66-5.67Google Scholar). Labeled RNA was purified on 1.5-mm 15% (w/v) polyacrylamide gels containing 7 m urea. RNA was eluted from the appropriate gel fragment by incubation in 0.1 m Tris/HCl, pH 8, 100 mm NaCl, 1 mm EDTA, 0.1% (w/v) SDS for 12 h at 37 °C. RNA was precipitated by the addition of 3 volumes of absolute ethanol. The pellet was collected by centrifugation, rinsed with absolute ethanol, and dried, before being resuspended in 50 μl of sterile water. MetJ repressor, purified as described (22He Y.-Y. McNally T. Manfield I. Navratil O. Old I.G. Phillips S.E.V. Saint-Girons I. Stockley P.G. Nature. 1992; 359: 431-433Crossref PubMed Scopus (29) Google Scholar,23.Parsons, I. D., Kinetic Studies of the E. coli Methionine Repressor-Operator Interaction. Ph.D thesis, 1996, University of Leeds, UK.Google Scholar), was a gift from Isobel D. Lawrenson (University of Leeds). It was supplied as an ammonium sulfate precipitate and stored at 4 °C until required. Precipitates were dissolved in 100 mm sodium phosphate (pH 7) and dialyzed against this buffer, prior to use. The protein concentration was determined spectrophotometrically (22He Y.-Y. McNally T. Manfield I. Navratil O. Old I.G. Phillips S.E.V. Saint-Girons I. Stockley P.G. Nature. 1992; 359: 431-433Crossref PubMed Scopus (29) Google Scholar,23.Parsons, I. D., Kinetic Studies of the E. coli Methionine Repressor-Operator Interaction. Ph.D thesis, 1996, University of Leeds, UK.Google Scholar). RNA labeled at either the 5′- or the 3′-end (≈1 pg, ≈1 × 106 cpm) was digested with RNase T1 (Boehringer Mannheim), RNase A (Sigma), or RNase U2 (Sigma) in 20 μl of 50 mm Tris/HCl, pH 7.0, 50 mm KCl, containing 6 units of tRNA (type X-SA, Sigma). Digestions were carried out at 37 °C for 15 min. The cleaved products were analyzed on a denaturing 19% (w/v) polyacrylamide gel containing 7 m urea and visualized by autoradiography or phosphorimaging (Fujifilm BAS 1500). Quantitation was carried out using the PhosphorImager associated software, TINA (Raytest Isotopenmeβgeräte GmbH). Diethyl pyrocarbonate (DEPC) modification of adenosine and guanosine was performed essentially as described in Ref. 24Conway L. Wickens M. Methods Enzymol. 1989; 180: 369-379Crossref PubMed Scopus (36) Google Scholar. Approximately 6 pg (≈5 × 106 cpm) of 5′- or 3′-labeled RNA in either 200 μl of native buffer (50 mm Tris/HCl, pH 7.0, 50 mm KCl) or 200 μl of denaturing buffer (50 mmsodium acetate, pH 4.5, 1 mm EDTA) were modified with 5 μl of DEPC at either 37 °C for 30 min (native buffer) or 90 °C for 2.5 min (denaturing buffer). The reaction was stopped with 75 μl of 1 m sodium acetate, pH 4.5, and 750 μl of absolute ethanol. Following centrifugation the RNA pellet was dissolved in 200 μl of 0.3 m sodium acetate, pH 3.8, and precipitated with 600 μl of absolute ethanol. The precipitated RNA was rinsed with absolute ethanol and dried. DEPC-modified RNA was then cleaved at the modified nucleotide by treatment with aniline (24Conway L. Wickens M. Methods Enzymol. 1989; 180: 369-379Crossref PubMed Scopus (36) Google Scholar). The fragmented products were examined by denaturing gel electrophoresis, as above. RNA was chemically modified with either DEPC, hydrazine, or ethylnitrosourea. Adenosine and guanosine were modified with DEPC, as described above, using the denaturing buffer. Uridine bases were modified by dissolving an ethanol precipitate of labeled RNA (≈6 pg, ≈5 × 106 cpm) in 10 μl of hydrazine hydrate (24Conway L. Wickens M. Methods Enzymol. 1989; 180: 369-379Crossref PubMed Scopus (36) Google Scholar). Following incubation on ice for 10 min, the reaction was stopped by the addition of 200 μl of 0.3 m sodium acetate, pH 3.8, and 750 μl of absolute ethanol. The RNA pellet was re-precipitated, as described above, and dissolved in 15 μl of water. RNA phosphate ethylation was carried out with ethylnitrosourea (25Vlassov V.V. Giegé Ebel J.-P. Eur. J. Biochem. 1981; 119: 51-59Crossref PubMed Scopus (93) Google Scholar). About 6 pg of labeled RNA (≈5 × 106 cpm) in 0.3 msodium cacodylate, pH 8.0, 2 mm EDTA, was treated with 4 μl of ethylnitrosourea (as a saturated ethanolic solution) at 80 °C for 2 min. The reaction was stopped by the addition of 3 μl of 3 m sodium acetate, pH 6.0, and 100 μl of absolute ethanol. After centrifugation, the RNA pellet was resuspended in 20 μl of 0.3 m sodium acetate, pH 6.0, 20 mmEDTA, and reprecipitated with 100 μl of absolute ethanol. The RNA pellet was rinsed with absolute ethanol, dried, and resuspended in 15 μl of water. The modified RNA (2–4 pg) was heated at 90 °C for 2 min, then placed at 37 °C for 15 min prior to its addition to 50 mm Tris/HCl, pH 7.0, 50 mm KCl, 10 units of RNAguard (Amersham Pharmacia Biotech), in the presence or absence of MetJ repressor (20 μm, dimer concentration) (final volume 20 μl). RNA binding does not require AdoMet (in contrast to the binding of Met box DNA) and so this was omitted from both the incubation buffer and the gel. The samples were incubated at 37 °C for 10 min, after which 5 μl of 50 mm Tris/HCl, pH 7.0, 50 mm KCl, 50% (w/v) glycerol was added. Gel retardation of the RNA was achieved by electrophoresis on a 1.5-mm-thick 10% (w/v) polyacrylamide gel, buffered with 10 mm sodium phosphate, pH 7.0. The samples were briefly run into the gel at 300 V, and the gels then were run at 100 V using 100 mm sodium phosphate (pH 7.0) running buffer. Following electrophoresis the appropriate bands were excised, and the RNA was recovered by elution as described above. 25 units of tRNA (type X-SA, Sigma) was added before the RNA was precipitated. Both DEPC- and hydrazine-modified RNAs were then cleaved at the modified nucleotides, using aniline, as described above. Ethylated RNA was cleaved with 0.1 m Tris/HCl, pH 9.0 (25Vlassov V.V. Giegé Ebel J.-P. Eur. J. Biochem. 1981; 119: 51-59Crossref PubMed Scopus (93) Google Scholar). The cleaved products were visualized by denaturing gel electrophoresis as described above. Filter binding assays were performed as described previously (6Parsons I.D. Persson B. Mekhalfia A. Blackburn G.M. Stockley P.G. Nucleic Acids Res. 1995; 23: 211-216Crossref PubMed Scopus (60) Google Scholar). The MetJ protein was serially diluted in 50 mm Tris/HCl, pH 7, 50 mm KCl and incubated with approximately 20 nm RNA for 10 min at 37 °C. Samples were filtered through 0.45-μm nitrocellulose filters (Whatman) and rinsed with 2 × 400 μl of binding buffer. Following drying, 5 ml of Ecoscint A (National Diagnostics) was added, and the samples were counted in a liquid scintillation counter. TheK d was estimated as the protein concentration at which half-maximal binding of the RNA occurred. Circular dichroism spectra were obtained using a Jobin Yvon CD6 Dichrograph with the cell (path length 10 mm) thermostatted at 37 °C, unless stated otherwise. Samples (0.15 ml) were prepared in 50 mm Tris/HCl, pH 7.0, 50 mm KCl. An RNA concentration of 1 μm was used, with the protein concentrations quoted in the legend of Fig. 9. Cross-linking of the MetJ repressor to RNA containing 4-thiouridine was carried out as described previously (26McGregor A. Rao M.V. Duckworth G. Stockley P.G. Connolly B.A. Nucleic Acids Res. 1996; 24: 3173-3180Crossref PubMed Scopus (35) Google Scholar). Samples containing 7.5 μm MetJ repressor dimer and approximately 50 nm radiolabeled oligoribonucleotide in 50 mm Tris/HCl, pH 7, 50 mm KCl, were incubated at 37 °C for 10 min prior to irradiation, at 350 nm, for 10 min. The oligoribonucleotide shown in Fig. 1 A was used, but with the uridines at positions 20, 23, 35, and 36 replaced with 4-thiouridine. Potential protein-RNA cross-links were analyzed by SDS-polyacrylamide gel electrophoresis. The sequence and the predicted secondary structure of the SELEX RNA ligand used in this study is shown in Fig. 1. It consists of a stem-loop (terminal loop) and an internal loop (27Zuker M. Methods Enzymol. 1989; 180: 51-59Crossref PubMed Scopus (1022) Google Scholar). To test this prediction experimentally, we have mapped the RNA ligand using ribonucleases and with DEPC. As shown in Fig.2, the terminal loop was most sensitive to cleavage by the single-strand specific nucleases RNase A ((C/U)-specific) and RNase U2 (A-specific). A number of nucleotides within and directly adjacent to the internal loop were also cleaved by the nucleases, but to a lesser degree than those in the terminal loop. These include A6/U7/A8 (5′-labeled strand) and C33/C38/C41 (3′-labeled strand). A few internal loop nucleotides, which would be expected to be cleaved, if single-stranded, were either not affected or were very poorly cut by the nucleases examined. Nucleotides in this category were C9/U10/C11 (5′-labeled strand) and A34/U35/U36/A39(3′-labeled strand). The nucleotide C29, which is predicted to be in a 2-nucleotide bulge in the main stem, was also susceptible to cleavage by RNase A. Consistent with most of the guanosines being double-stranded, RNase T1 (G-specific) did not cleave well, including position G37 within the internal loop. Comparison of DEPC modification (which modifies the N7 position of adenosine and, to a lesser extent, that of guanosine) of the RNA at 37 °C and 90 °C showed marked differences, with greater modification under denaturing as compared with native conditions, for two of the three adenosines (A31/A42), predicted to be in double-stranded regions (Fig. 2). The four adenosines (A6/A8/A34/A39) which are predicted to be in the internal loop, were also modified to a greater degree in the denatured oligonucleotide compared with the native one. In contrast, little difference in reactivity was observed for the two adenosines (A21/A22) in the terminal loop. The nucleotide A24 also behaved in this manner. Although predicted to be in a double-stranded region, this nucleotide is immediately adjacent to the terminal loop. The results are summarized in Fig. 3 and are in broad agreement with the predicted structure. The terminal loop, which closes the stem-loop, has properties consistent with its being single-stranded. The regions predicted to be double-stranded map as a duplex when probed with nucleases or DEPC. The most interesting region is the internal loop. A number of nucleotides within this region, that would be expected to be ribonuclease-sensitive, were either not cleaved or were cut significantly more slowly than those in the terminal loop. Similarly, several nucleotides have a diminished reactivity to DEPC in the native compared with the denatured RNA. This suggests that the internal loop cannot be considered as single-stranded but must contain some degree of stable secondary structure, limiting the accessibility of both ribonucleases and DEPC. It is conceivable that some of the bases on opposite sides of the internal loop form non-canonical base pairs. In addition bases on the same strand may be strongly stacked. Interactions between the bases in the RNA ligand and the MetJ repressor have been determined by binding-interference analysis. DEPC has been used to probe the role of adenosine and guanosine, and hydrazine has been utilized for uridine. The results for DEPC are shown in Fig. 4. To map the entire RNA sequence, it was necessary to use both 3′- and 5′-end labeling. A similar quality autoradiogram (not shown) was obtained using hydrazine. Scans of both the DEPC and hydrazine autoradiograms are shown in Fig. 5, and the results are summarized in Fig. 6. Both DEPC and hydrazine caused notable interference at a number of bases. Modification of bases within, and directly adjacent to, the internal loop significantly reduced the binding of the RNA ligand to MetJ. Modification of the eight bases in and around the terminal loop had no effect on binding. Modification of the bases in the intervening stem that connects the internal and the terminal loops had a mildly detrimental effect on binding. This strongly suggests that it is the internal loop region of the RNA ligand that is responsible for tight binding to the MetJ repressor.Figure 5Densitometry of the interference data found for MetJ using DEPC and hydrazine-modified RNA. A, 3′-labeled RNA modified with DEPC; B, 5′-labeled RNA modified with DEPC; C, 3′-labeled RNA modified with hydrazine; D, 5′-labeled RNA modified with hydrazine.Black, RNA that was gel-shifted by MetJ; red, RNA that failed to shift in the presence of MetJ protein; blue, RNA run on the gel in the absence of protein.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Summary of the interference analysis data found for MetJ using DEPC and hydrazine-modified RNA. Interference by DEPC and hydrazine are denoted by circles andtriangles, respectively. Two symbolsrepresent strong interference and one symbolsignifies interference. Strong interference and interference are defined by modification interference indexes of greater than 4 and between 2 and 4, respectively (14Convery M.A. Roswell S. Stonehouse N.J. Ellington A.D. Hirao I. Murray J.B. Peabody D.S. Phillips S.E.V. Stockley P.G. Nat. Struct. Biol. 1998; 5: 133-139Crossref PubMed Scopus (118) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The importance of the internal loop was confirmed using two truncated RNA ligands. The deletions remove (a) the terminal loop and 2 base pairs from the stem and (b) the terminal loop, 5 base pairs from the stem, and the two single stranded cytosines (Fig. 1). In both cases the deleted bases were replaced by a hexaethylene glycol chain to substitute as a hairpin loop (28Durand M. Chevrie K. Chessignol M. Thuong N.T. Maurizot J.C. Nucleic Acids Res. 1990; 18: 6353-6359Crossref PubMed Scopus (124) Google Scholar). The binding affinity of the two shortened RNA molecules to the MetJ repressor were determined using a filter binding assay (Fig. 7). Neither deletion markedly affected the binding of the RNA to MetJ.K d values (assessed as the protein-dimer concentration at which one-half of the counts were retained on the filter) of between 2 and 4 ± 0.5 × 10−7m were seen for both the deletions and the parent RNA ligand. A control RNA, consisting of the first 21 nucleotides of the parent RNA, did not bind to MetJ. These results clearly demonstrate that the initial, synthetic SELEX RNA fragment used does in fact bind tightly to MetJ as expected from the original SELEX experiment.2 Furthermore, we confirm that the internal loop of the RNA ligand encompasses the high affinity site for the MetJ repressor, in agreement with the binding-interference analysis. Although the terminal loop is not directly involved in binding it is necessary to allow the reversal of the RNA strand and hence the formation of the internal loop. Ethylnitrosourea, which ethylates the non-esterified oxygen atoms of backbone phosphates in nucleic acids, was used to probe the role of the RNA phosphate groups in binding to the MetJ repressor. No single phosphate was found to be critical for interaction with the MetJ repressor, i.e. there was no phosphate group, which when ethylated, strongly inhibited the binding of MetJ. This is shown in Fig. 8, which shows scans of the interference autoradiograms. The role of RNA phosphates in binding to MetJ has been further evaluated by the determination of K d values at various salt concentrations using a filter binding assay (data not shown). If interactions between negatively charged phosphates and positively charged amino acid side chains are important, theK d would be expected to increase with increasing ionic strength (29Lohman T.M. Mascotti D.P. Methods Enzymol. 1992; 212: 400-424Crossref PubMed Scopus (143) Google Scholar). In 10 mm Tris/HCl, pH 7.0, with KCl concentrations of 10, 25, 50, and 100 mm, identicalK d values of 1.2 ± 0.3 × 10−7m were obtained. The dissociation constant was marginally higher at KCl concentrations of 150 and 200 mm, 2.3 ± 0.5 × 10−7 and 6.3 ± 1 × 10−7m, respectively. Interestingly, although the data at 100–200 mm salt fitted a typical protein-ligand binding isotherm, those at 10–50 mm KCl were notably steeper, suggesting that some form of cooperativity occurs. One possible explanation is that MetJ dimers may dissociate at low concentrations in low salt buffers. Although it has been shown that thermally denatured MetJ protein will readily reassociate (30Johnson C.M. Cooper A. Stockley P.G. Biochemistry. 1992; 31: 9717-9742Crossref PubMed Scopus (38) Google Scholar), we have no evidence for this dissociation. However, the related Arc repressor from bacteriophage P22 does dissociate into unfolded monomers with a dissociation constant of 5 × 10−9m (31Brown M.B. Bowie J.U. Sauer R.T. Biochemistry. 1990; 29: 11189-11195Crossref PubMed Scopus (58) Google Scholar, 32Burgering J.M.M. Hald M. Boelans R. Breg J.N. Kaptein R. Biopolymers. 1995; 35: 217-226Crossref PubMed Scopus (19) Google Scholar). Alternatively, salt-dependent conformational changes of the RNA ligand could be involved, although the salt concentrations examined here had no effect upon the circular dichroism spectra of the RNA (not shown). Steep binding curves have been observed for the binding of MetJ protein to its DNA target (3Phillips S.E.V. Manfield I. Parsons I.D. Davidson B.E. Rafferty J.B. Somers W.S. Margarita D. Cohen G.N. Saint-Girons I. Stockley P.G. Nature. 1989; 341: 711-715Crossref PubMed Scopus (96) Google Scholar). Because of these complexities caution is needed in the interpretation of how the K d varies with ionic strength. Nevertheless, the minor effect of salt concentration upon the affinity of the RNA ligand binding to MetJ agrees with the lack of phosphate ethylation interference. Both experiments suggest that interactions between the phosphates of the RNA ligand and the MetJ play a minor role in binding. Circular dichroism data (Fig.9) are consistent with the RNA being largely double-stranded and in an A-form conformation. The spectrum of the free RNA shows a large positive Δε at 263 nm and a small negative peak at 295 nm, typical of an A-form helix. Heating the RNA caused a decrease in the intensity of the main peak and a shift to 271 nm, consistent with the conversion of the RNA to a single strand (33Gray D.M. Su-Hwi H. Johnson K.H. Methods Enzymol. 1995; 246: 19-35Crossref PubMed Scopus (243) Google Scholar). This is consistent with the mapping data presented above, which show that the internal loop cannot be considered single-stranded, but must have some element of structure. Thus, with the exception of the single-stranded terminal loop, almost all of the RNA ligand appears to exist as an A-form duplex. However, the exact structure of the internal loop may deviate somewhat from classical A-duplex parameters. When the RNA ligand binds to the MetJ protein, there is hardly any change in the circular dichroism spectrum of the nucleic acid (Fig. 9), especially above 240 nm where almost all (≥90%) of the intensity is due to the nucleic acid. This suggests that there are no large conformational changes to the RNA after binding to the protein. It has previously been shown that both oligodeoxyribonucleotides containing 4-thiothymidine and oligoribonucleotides containing 4-thiouridine can be used to photocross-link DNA- and RNA-binding proteins (26McGregor A. Rao M.V. Duckworth G. Stockley P.G. Connolly B.A. Nucleic Acids Res. 1996; 24: 3173-3180Crossref PubMed Scopus (35) Google Scholar, 34Nikiforov T.T. Connolly B.A. Nucleic Acids Res. 1992; 20: 1209-1214Crossref PubMed Scopus (53) Google Scholar). We had hoped to use this approach to map the regions of MetJ in contact with the RNA ligand. Unfortunately, substitution of several uridines by 4-thiouridine, in the oligoribonucleotide shown in Fig. 1 A, and subsequent 350 nm irradiation of these oligoribonucleotides, in the presence of MetJ, failed to generate any cross-links (data not shown). Considering the data above, this is hardly surprising for the uridines (positions 20 and 23) in the terminal loop. The failure of 4-thiouridines, within the internal loop (positions 35 and 36), to cross-link was disappointing, particularly as modification of uridines 35 and 36, with hydrazine, interferes with MetJ binding (Figs. 5 and 6). However, we have previously shown that the proximity of 4-thiouridine to a protein is not necessarily sufficient for cross-linking to occur (26McGregor A. Rao M.V. Duckworth G. Stockley P.G. Connolly B.A. Nucleic Acids Res. 1996; 24: 3173-3180Crossref PubMed Scopus (35) Google Scholar). Filter binding (not shown) demonstrated that replacement of uridines 20, 23, 35, and 36 with 4-thiouridine either did not affect binding or reduced it to a small extent, such that significant amounts of MetJ-RNA complex was formed at the concentrations used for photocross-linking. Although there is a great deal of interest in RNA ligands selected to bind particular target molecules with high affinity, there is, as yet, little structural information on the nature of the RNA-target complexes. In the last few years several structures have been determined for selected RNAs bound to small molecular weight ligands such as ATP, using NMR spectroscopy (35Heuss H.A. Nat. Struct. Biol. 1997; 4: 597-600Crossref PubMed Scopus (15) Google Scholar). More recently the first high resolution crystal structure for a selected RNA-protein complex has been determined (14Convery M.A. Roswell S. Stonehouse N.J. Ellington A.D. Hirao I. Murray J.B. Peabody D.S. Phillips S.E.V. Stockley P.G. Nat. Struct. Biol. 1998; 5: 133-139Crossref PubMed Scopus (118) Google Scholar). Additionally, low resolution methods, mainly interference and protection modification combined with base substitutions, have been used to map the interfaces between proteins and selected, high affinity, RNA ligands (17Green L. Waugh S. Binkley J.P. Hostomska Z. Hostomsky Z. Tuerk C. J. Mol. Biol. 1995; 247: 60-68Crossref PubMed Scopus (48) Google Scholar).