6772
Biochemistry 2003, 42, 6772-6783
Monitoring RNA Base Structure and Dynamics Using Site-Directed Spin Labeling† Peter Z. Qin,‡,§,| Ka´lma´n Hideg,⊥ Juli Feigon,§ and Wayne L. Hubbell*,‡,§ Jules Stein Eye Institute and Department of Chemistry and Biochemistry, UniVersity of California, Los Angeles, Los Angeles, California 90095, Department of Chemistry, UniVersity of Southern California, LJS-251, 840 Downey Way, Los Angeles, California 90089-0744, and Institute of Organic and Medical Chemistry, UniVersity of Pe´ cs, P.O. Box 99, H-7643 Pe´ cs, Hungary ReceiVed NoVember 21, 2002; ReVised Manuscript ReceiVed April 14, 2003
ABSTRACT: Site-directed spin labeling utilizes site-specific attachment of a stable nitroxide radical to probe the structure and dynamics of macromolecules. In the present study, a 4-thiouridine base is introduced at each of six different positions in a 23-nucleotide RNA molecule. The 4-thiouridine derivatives were subsequently modified with one of three methanethiosulfonate nitroxide reagents to introduce a spin label at specific sites. The electron paramagnetic resonance spectra of the labeled RNAs were analyzed in terms of nitroxide motion and the RNA solution structure. At a base-paired site in the RNA helix, where the nitroxide has weak or no local interactions, motion of the nitroxide is apparently dominated by rotation about bonds within the probe. The motion is similar to that found for a structurally related probe on helical sites in proteins, suggesting a similar mode of motion. At other sites that are hydrogen bonded and stacked within the helix, local interactions within the RNA molecule modulate the nitroxide motion in a manner consistent with expectations based on the known structure. For a base that is not structurally constrained, the mobility is higher than at any other site, presumably due to motion of the base itself. These results demonstrate the general utility of the 4-thiouridine/methanethiosulfonate coupling method to introduce nitroxide spin labels into RNA and the ability of the resulting label to probe local structure and dynamics.
RNA is a versatile molecule that plays multiple roles in gene expression and acts as an information carrier, catalyst, and regulator (1). Many aspects of RNA function depend on its ability to form compact and complex three-dimensional structures. During the past 5 years, there has been an explosion of high-resolution RNA structures, increasing the size of the database of known RNA structures (2). However, to elucidate structure-function relationships, it is necessary to go beyond the static structure and understand the dynamics of the molecule in solution. For this purpose, it is essential to develop spectroscopic tools that provide information on both fluctuations of the structure at equilibrium and conformational changes. Picosecond to nanosecond motions in RNA bases have been studied by NMR spectroscopy (35). Conformational rearrangements of RNA loops in the † Research reported here was supported by NIH Grant EYO5216 (W.L.H.), the Jules Stein Professor Endowment (W.L.H.), the Bruce Ford Bundy and Anne Smith Bundy Foundation (W.L.H.), NSF Grant MCB-9808072 (J.F.), NIH Grant R01 GM 37254 (J.F.), the Hungarian National Research Foundation (OTKA T034307 to K.H.), and the startup fund from the University of Southern California (P.Z.Q.). P.Z.Q. was a DuPont Pharmaceutical Fellow of the Life Sciences Research Foundation. * To whom correspondence should be addressed at the Jules Stein Eye Institute, UCLA School of Medicine, 100 Stein Plaza, Los Angeles, CA 90095-7008. Tel: (310) 206-8830. Fax: (310) 794-2144. E-mail:
[email protected]. ‡ Jules Stein Eye Institute, University of California, Los Angeles. § Department of Chemistry and Biochemistry, University of California, Los Angeles. | Department of Chemistry, University of Southern California. ⊥ Institute of Organic and Medical Chemistry, University of Pe´cs.
microsecond to millisecond regime have been studied by both NMR spectroscopy (6) and fluorescence spectroscopy (7). Conformational changes of large RNA molecules on the millisecond or slower time scales have been probed by fluorescence spectroscopy (8-12) and chemical modification footprinting (13, 14). These studies reveal an enormous complexity in RNA dynamics and show that there is a general correlation between RNA dynamics, structure, and function. The technique of SDSL1 has been developed to study structure and dynamics in proteins (15-21). The basic strategy of SDSL in proteins involves the substitution of a cysteine for the native residue, followed by modification of the reactive SH group with a selective paramagnetic nitroxide reagent. The most commonly employed reagent is a methanethiosulfonate derivative that generates a disulfide-linked nitroxide side chain. Analyzing the EPR spectrum of the labeled protein yields information on three aspects of interest: (1) the dynamics of the nitroxide (i.e., the overall rotational motion of the nitroxide); (2) the collision rate between the nitroxide and a freely diffusing paramagnetic agent, a quantity proportional to the solvent accessibility; and (3) the distance between the nitroxide and another paramagnetic species fixed in the structure. Methods have 1
Abbreviations: buffer A, 10 mM sodium phosphate, pH 6.8; DTT, EPR, electron paramagnetic resonance; MMTS, methylmethanethiosulfonate; MOPS, 3-(N-morpholino)propanesulfonic acid; SDSL, site-directed spin labeling; T4L, T4 lysozyme; TEMPOL, 1-oxy-4-hydroxy-2,2,6,6-tetramethylpiperidine; TLC, thin-layer chromatography. DL-dithiothreitol;
10.1021/bi027222p CCC: $25.00 © 2003 American Chemical Society Published on Web 05/15/2003
Monitoring RNA Base Structure and Dynamics with SDSL
Biochemistry, Vol. 42, No. 22, 2003 6773
been established to extract protein structural and dynamic information from these parameters. For example, the motion of the nitroxide side chain can provide information on backbone dynamics (21, 22) and conformational changes (19), while the sequence dependence of solvent accessibility identifies regular secondary structure and features of the tertiary fold (23). The interspin distance between two nitroxides, or between a nitroxide and a fixed paramagnetic metal ion, provides direct structural data (24-28). SDSL allows one to deduce key structural features of a protein at the backbone level and, more importantly, to follow structural changes by monitoring the time-dependent variation of the spectral parameters of a spin label. The scope and capabilities of the method have been demonstrated in studies of many proteins, especially membrane or membraneassociated proteins that are difficult to tackle with other techniques. With the advantage of being able to gain information from a small amount of sample (∼100 pmol) in solution conditions, SDSL should also be an ideal technique for studying RNA structure and dynamics. SDSL has only recently been applied to study RNA (2932). Because SDSL is a method that relies on external reporter groups, efficient attachment of the spin label to RNA is critical. Labeling at RNA backbone locations has been achieved by introducing a site-specific phosphorothioate group and subsequent reaction with an R,β-unsaturated iodomethane derivative of a nitroxide (31). Spin labels have also been attached to the 5′ terminus of RNA by taking advantage of a 5′-phosphorothioate (29). Substitution of specific 2′-hydroxyl groups with amine groups allows the attachment of nitroxides at specific sugar positions (30). Attachment of nitroxides to modified RNA bases using iodoacetamide reagents has also been reported (33). Currently, most SDSL studies of RNA have focused on deriving RNA structural information from the motion of the nitroxide (30-32). The overall motion of a nitroxide in a macromolecule is determined by three dynamic modes: (1) the rotation of the entire molecule (characterized by correlation time τR); (2) torsional oscillations about bonds that connect the nitroxide moiety to the macromolecule; and (3) structural fluctuations within the macromolecule. Although τR does not contain site-specific information, changes of τR can be used to probe RNA/RNA interactions in solution (31). On the other hand, the other motions reflect the RNA local environment and dynamics. The point of attachment and the structure of the probe dictate the type of information that can be obtained. In RNA, the nucleobase and the sugar ring are connected by a glycosidic bond, and in general, the base and the sugar have different dynamic characteristics. To probe base dynamics, it is therefore important to develop methods to attach nitroxide probes directly to the RNA base. The naturally occurring 4-thiouridine modification, which contains a reactive thiol group and can be incorporated into RNA both enzymatically or via chemical synthesis, provides an attractive target for spin labeling. An iodoacetamide spin label derivative has been employed to attach a spin label to a 4-thiouridine base for the purpose of obtaining distance constraints in NMR studies of an RNA/protein complex (33). However, the sensitivity of the spin label to local RNA structure and dynamics has not been investigated.
The goals of the present study are twofold: (1) to devise a method to attach nitroxide spin labels to specific base positions within an RNA molecule and (2) to investigate the correlation between the nitroxide motion and the RNA base structure and dynamics. For attachment of spin labels to RNA, it is shown that thiol-reactive methanethiosulfonate nitroxide reagents, which have been extensively investigated in protein studies (15), selectively derivatize a site-specifically substituted 4-thio-U base (Figure 1A). Thus, the spinlabeled 4-thio-U base itself is employed as a probe, and evidence is presented to show that the motion of the nitroxide reflects both local RNA structure and the dynamics of the base to which it is attached. To explore the utility of the probes as monitors of RNA structure and dynamics, a 23-nucleotide RNA molecule (Figure 1B), which contains the 11-nucleotide GAAA tetraloop receptor motif (34) capped by an UUCG terminal loop, was selected as a model. In keeping with previous literature, this RNA is hereafter referred to as “TLR” (35). Although there are less complex structures available, TLR was selected because the solution structure has been obtained by NMR (35) and the dynamics of the UUCG loop investigated (3). The RNA provides several different arrangements of U nucleotides (Watson-Crick paired, nonWatson-Crick paired, unpaired) to test the utility of the spin label in probing different environments. In addition, the GAAA tetraloop receptor motif is a component of one of the most frequently occurring RNA tertiary interactions: the GAAA tetraloop/receptor interaction (34). Spin labeling studies reported here provide a foundation for future investigation of the GAAA tetraloop/receptor interaction using the SDSL method. EPR spectral analyses show that at a base-paired site in the RNA helix, where the nitroxide has weak or no local interactions, the motion of the nitroxide in label Ra (Figure 1A) is constrained and apparently dominated by the rotation about bonds within the nitroxide probe. The motion is essentially identical to that found for a similar label on helical surface sites in proteins, suggesting a similar origin. At other base-paired sites, local interactions within the RNA molecule modulate this motion in a manner consistent with expectations based on the known structure. Finally, at a previously identified non-base-paired site in the UUCG tetraloop, the nitroxide shows the highest mobility observed, presumably reflecting the motion of the base itself. MATERIALS AND METHODS RNA Preparation. The sequence of TLR is 5′GGCCUAAGACUUCGGUUAUGGCC. TLR molecules containing singly substituted 4-thiouridine at positions 5, 11, 12, 16, 17, and 19 (numbering from 5′ terminus) are designated 4-thioUx-TLR, where x indicates the position of substitution. The 4-thio-Ux-TLR molecules were obtained from Dharmacon Research, Inc. (Lafayette, CO) and deprotected according to protocols provided by the vendor. For NMR studies, TLR was generated enzymatically as reported (35). All RNAs were purified by denaturing polyacrylamide gel electrophoresis. Synthesis of Spin-Labeling Reagents. General. Melting points were determined on a Boetius micro-melting point apparatus and are uncorrected. Elemental analyses (C, H,
6774 Biochemistry, Vol. 42, No. 22, 2003
Qin et al.
FIGURE 1: (A) 4-Thiouridine labeling scheme. An uridine base is substituted by 4-thiouridine during chemical synthesis and subsequently labeled with methanethiosulfonate derivatives a, b, or c to generate the corresponding labels Ra, Rb, or Rc. (B) Sequence and secondary structure of the TLR molecule. The bases are represented by open rectangles and numbered from 5′ to 3′. Dashed lines between bases represent hydrogen bonds. Small black rectangles between bases represent stacking interactions. The six uridines within the TLR are shown in red. (C) Absorbance spectra of wild-type and modified TLR. Representative spectra between 220 and 400 nm are shown for TLR molecules with modifications at U16, and the difference spectra between wild-type and modified TLR are shown in the corresponding insets.
N, and S) were performed on EA 1110 CHNS elemental analyzer, and the bromine was analyzed tritrimetrically by Scho¨niger’s method. IR spectra were recorded on a Zeiss Specord 75. Mass spectra were recorded on a Finnigan Automass Multi instrument. The thiol-reactive reagent a [1-oxyl-3-(methanesulfonylthiomethyl)-2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrole] was synthesized as reported (36), and the thiol-reactive reagent b [1-oxyl-3-(methanesulfonylthiomethyl)-2,5-dihydro-2,2,4,5,5-pentamethyl-1H-pyrrole] was synthesized as previously reported (37). Reagent c was prepared according to Scheme 1. 1-Oxyl-3-(1-bromoethyl)-2,5-dihydro-2,2,5,5-tetramethyl1H-pyrrole (2). To a stirred solution of 1-oxyl-3-(2-hydroxyethyl)-2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrole (1) (920 mg, 5.0 mmol, prepared as described in ref 38) and Et3N (555 mg, 5.5 mmol) in CH2Cl2 (30 mL) was added methanesulfonyl chloride (920 mg, 5.5 mmol) dropwise at 0 °C. The mixture was stirred at room temperature for 1 h.
Scheme 1
The organic phase was washed with brine (10 mL), separated, dried (MgSO4), filtered, and evaporated to yield the corresponding mesylate. The crude mesylate and LiBr (434 mg, 5.0 mmol) were stirred and refluxed in acetone (20 mL) for 1 h. Acetone was evaporated, and the residue was dissolved in methylene dichloride (20 mL) and washed with brine (15 mL). The organic phase was dried, filtered, evaporated, and purified with flash column chromatography on silica gel (hexane/Et2O) to give 1-oxyl-3-(1-bromoethyl)-2,5-dihydro2,2,5,5-tetramethyl-1H-pyrrole (2) as a deep yellow oil: 890 mg (72%). Anal. Calcd for C10H17BrNO (247.15): C, 48.60;
Monitoring RNA Base Structure and Dynamics with SDSL
Biochemistry, Vol. 42, No. 22, 2003 6775
H, 6.93; N, 5.67; Br, 32.33. Found: C, 48.50; H, 7.10; N, 5.50; Br, 32.50. MS m/z (%): 248/246 (M+, 10), 218/216 (4), 167 (19), 152 (84), 137 (100). 1-Oxyl-3-(1-methanesulfonylthioethyl)-2,5-dihydro-2,2,5,5tetramethyl-1H-pyrrole (3). The above bromide 2 (247 mg, 1.0 mmol) and NaSSO2CH3 (202 mg, 1.5 mmol) in aqueous acetone (8:2) (10 mL) was cautiously heated to 50 °C, and the mixture was monitored by TLC. After consumption of starting material (30 min), the solvent was removed by evaporation, and the residue was extracted with CHCl3 (2 × 30 mL). The organic phase was separated, dried (MgSO4), filtered, and evaporated, and the residue was purified by flash chromatography on silica gel (hexane/ethyl acetate) to yield the title compound 3 as an orange solid: 181 mg (65%); mp 116 °C. Anal. Calcd for C11H20NO3S2 (278.40): C, 47.46; H, 7.24; N, 5.03; S, 23.03. Found: C, 47.70; H, 7.10; N, 5.20; S, 23.30. MS m/z (%): 278 (M+, 21), 264 (8), 248 (4), 169 (100), 152 (80). Labeling of 4-Thiouridine DeriVatiVes. RNA containing singly substituted 4-thiouridine (1-50 µmol in 100 µL) was treated for 30 min with 100 µM DTT in buffer A at room temperature. The reaction mix was then loaded onto a PD10 column (Amersham Biosciences) and eluted with buffer A. Fractions containing the RNA (∼2 mL) were reacted directly with the appropriate nitroxide reagent or MMTS (Toronto Research Chemicals, North York, Ontario, Canada) (final concentration 100 µM). After incubation at room temperature overnight, the excess reagent was removed by first passing it through a PD-10 column, followed by extensive washing using an Ultrafree 0.5 centrifugal filter (Millipore, Inc.). Labeled RNAs were stored in water at -20 °C to minimize the detachment of spin label from RNA due to the labile nature of the disulfide bond on the uridine heterocycle. Under conditions reported here, the labeled RNA can be stored at least 4 weeks, and dissociation of the label during room temperature measurement was slow (
