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With these comparisons in mind, kinetic oligonucleotide hybridization is a reasonable first step in monitoring the folding kinetics of a large RNA, especially if several useful probes can be identified. Then, in conjunction with other methods such as CD and SHRF, more detailed questions can be addressed that require increased time and structural resolution. Acknowledgments This work was supported by the Rita Allen Foundation and the Skaggs Institute for Chemical Biology. The authors wish to acknowledge Patrick Zarrinkar, who developed kinetic oligonucleotide hybridization during his thesis work, for his important contributions to this work and for critical comments on the manuscript.
[22] T i m e - R e s o l v e d S y n c h r o t r o n X - R a y F o o t p r i n t i n g a n d Its Application to RNA Folding B y CORIE Y . RALSTON, BIANCA SCLAVI, MICHAEL SULLIVAN, MICHAEL L . DERAS, SARAH A . WOODSON, M A R K R . CHANCE, a n d MICHAEL BRENOWITZ
Introduction The ability of RNA molecules to form uniquely folded, compact tertiary structures is critical to their biological function. Determining the mechanisms of RNA folding is essential to understanding their binding and catalytic functions in diverse cellular processes such as translation and splicing. In particular, catalytic RNAs derive their enzymatic ability from the formation of discrete tertiary structures composed of multiple domains. 1-3 Individual RNA domains can fold on timescales ranging from milliseconds to minutes. 4 Characterizing RNA folding pathways and deducing their common features is an important challenge that will require the concomitant application of a variety of structural and functional techniques. Nucleic acid footprinting assays map the solvent accessible surface of DNA or RNA by probing their protection from nucleases or modifying reagents. The development of quantitative footprinting protocols allows 1 T. R. Cech, in "The RNA World," (R. F. Gesteland and J. F. Atkins, eds.), pp. 239-269. Cold Spring Harbor Laboratory Press, New York, 1993. 2 T. R. Cech, D. Herschlag, J. A. Piccirilli, and A. M. Pyle, J. Biol. Chem. 267(25), 17479 (1992). 3 T. R. Cech and D. Herschlag, Nucleic Acids Mol. Biol. 10, 1-7 (1996). 4 For a review, see D. E. Draper Nature Struct. Biol. 3, 397 (1996).
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isotherms and kinetic progress curves to be obtained for individual sites within a nucleic acid. 5-7 Protocols for quench-flow DNase I footprinting established the feasibility of conducting footprinting kinetics studies on the millisecond timescale. 6 Since the first DNase I footprinting studies of DNA-protein interactions, 8 a wide range of enzymatic and chemical cleavage agents has been successfully applied to the study of R N A and D N A structure and dynamics and their interaction with proteins. Of the plethora of nucleases available to investigators, perhaps none is more widely used than the hydroxyl radical ('OH). 9'1° The key advantage to -OH footprinting is that the radicals are small enough to provide sensitivity to conformational changes with single base pair resolution. In footprinting assays, -OH can be generated through the radiolysis of water, 11'12or by catalysts such as Fe-EDTA 9 or peroxonitrous acid. 13 Synchrotron X-ray "footprinting" is a new technique that allows time-resolved structural analysis of conformational changes of nucleic acids using .OH generated by the passage of a bright synchrotron x-ray beam through water. The solvent-accessible surface of a nucleic acid is probed by t h e . O H mediated cleavage of the phosphodiester backbone. The extremely high brightness of the synchrotron X-ray beam allows millisecond X-ray exposures to be used in footprinting experiments. Thus, the entire solventaccessible surface of a nucleic acid can be mapped with single base resolution, on timescales as short as milliseconds. The technique has been successfully applied to the study of the Tetrahymena thermophila group I intron, yielding information on folding rate
5 M. Brenowitz, D. F. Senear, M. A. Shea, and G. K. Ackers, Methods Enzymol. 130, 132 (1986). 6 M. Hsieh and M. Brenowitz, Methods Enzymol. 274, 478 (1996). 7 D. Strahs and M. Brenowitz J. Mol. Biol. 244, 494 (1994). 8 D. J. Galas and A. Schmitz, Nucleic Acids Res. 5, 3157 (1978); A. Schmitz and D. J. Galas, Nucleic Acids Res. 6, 111 (1979). 9 T. D. Tullius, B. A. Dombroski, M. E. Churchill, and L. Kam, Methods Enzymol. 155, 537 (1987); T. D. Tullius and B. A. Dombraski, Proc. Natl. Acad. Sci. U.S.A. 83, 5469 (1986); W. J. Dixon, J. J. Hayes, J. R. Levin, M. F. Weidner, B. A. Dombroski, and T. D. TuUius Methods Enzymol. 208, 380 (1991). 10D. C. Celander and T. R. Cech Science 251, 401 (1991); D. C. Celander and T. R. Cech, Biochemistry 29, 1355 (1990). 11M. A. Price and T. D. Tullius Methods Enzymol. 212, 194 (1992). 12j. Franchet-Beuzit, M. Spotheim-Maurizot, R. Sabattier, B. Blazy-Baudras, and M. Charlier Biochemistry 32, 2104 (1993). 13 p. A. King, V. E. Anderson, J. O. Edwards, G. Gustafson, R. C. Plumb, and J. W. Suggs, J. Am. Chem. Soc. 114, 5430 (1992); P. A. King, E. Jamison, D. Strahs, V. E. Anderson, and M. Brenowitz, Nude&Acids Res. 21, 2473 (1993).
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constants within all its major domains. 14,15 Synchrotron X-ray footprinting is currently being applied to the study of the structure and folding of other R N A molecules, the structure of DNA, D N A - p r o t e i n interactions, and protein foldingJ 6 An article has been published in this series describing the development of synchrotron .OH footprinting and its application to R N A folding. 17 In this article we report the advances that have been made in the application of synchrotron X-ray footprinting to the study of R N A folding at National Synchrotron Light Source (NSLS) beam line X-9A under the auspices of the Albert Einstein Center for Synchrotron Biosciences. The instrumentation and protocols that have been developed are applicable to other experimental systems and new X-ray footprinting facilities that might be established.
P h o t o n Flux a n d A b s o r p t i o n Calculation o f B e a m Flux
Beam line X-9A at the NSLS at Brookhaven National Laboratory is a bending magnet beam line producing white light over an energy range of 3-30 keV. The photon flux at the beam line can be calculated using bending magnet radiation curves and is dependent on the magnet strength and the energy and number of the electrons in the storage ring. TM Currently, the NSLS operates at two ring energies, 2.54 and 2.8 GeV. A t 2.54 GeV, the beam current decays from - 3 0 0 m A at the time of injection to - 1 5 0 m A over a 12-hr period. Similarly, the beam current decays from - 2 5 0 m A at injection to ~120 m A at the 2.8-GeV ring energy. The flux incident on the sample is obtained by multiplying the bending magnet radiation curve by the transmission curves for each absorber be-
x4B. Sclavi, S. Woodson, M. Sullivan, M. R. Chance, and M. Brenowitz, J. Mol. Biol. 266, 144 (1997). is B. Sclavi,M. Sullivan, M. R. Chance, M. Brenowitz,and S. A. Woodson, Science 279, 1940 (1998). 16M. R. Chance, B. Sclavi, S. Woodson, and M. Brenowitz, Structure 5, 865 (1997); M. R. Chance, M. Brenowitz, MI Sullivan, B. Sclavi,S. Maleknia, and C. Ralston, J. Synchr. Rad. 11, 7 (1998). 17B. Sclavi,S. A. Woodson, M. Sullivan,M. R. Chance, and M. Brenowitz,Methods Enzyrnol. 295, 379 (1998). 18Spectral curves for bend magnet radiation are available at the web page maintained by the Center for X-ray Optics at the Lawrence Berkeley Laboratory: http://www-cxro.lbl.gov/ optical_constants.
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Stopped-Flow
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tween the front end of the beam line and the sample. 19 In the current configuration of X-9A, two beryllium windows, 254 and 500/zm thick, cap each end of the "beam pipe," a tube under high vacuum (>10 -8 Torr) extending from the ring wall into the experimental hutch (Fig. 1). An aluminum coating on the exit window is 25/zm thick. After exiting the beam pipe, the photons travel through an air path of 40 cm before striking the exposure cell of the stopped-flow apparatus (Fig. 1). The radiation curve calculated for X-9A at 2.8-GeV operation and 250-mA beam current is shown in Fig. 2A (solid fine). Figure 2A also shows the calculated flux incident on a sample after passing through the two beryllium windows, the aluminum coating, and an air path of 40 cm (Fig. 2A, dash-dot fine). It 19Transmission curves were also obtained from the Center for X-Ray Optics web page; these transmission curves do not take into account scattering. However, comparison with transmission curves obtained by using the mass attenuation coefficient showed that scattering by water in the energy range of interest resulted in less than a 1% difference in the calculation of flux absorbed by the sample.
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can be seen that the beam line optics absorb more than 99% of the efflux from the ring and that the peak energy is shifted from - 5 to 10 keV. The flux transmitted through the sample is calculated by multiplying the flux incident on the sample by the transmission curve for a 1-mm water path length (Fig. 2A, dashed line). The difference between the incident flux and the transmitted flux is the amount absorbed by the sample (Fig. 2A, dotted line). Integration of the absorption curve gives the total number of photons per second absorbed by the sample. For a ring energy of 2.8
358 TECHNIQUES FOR MONITORING R N A CONFORMATION AND DYNAMICS [22] GeV and a beam current of 250 mA, 5.5 x 1014photons are absorbed per second by a 10-/zl sample of 7-mm 2 cross-sectional area, with the absorption maximum near 7.5 keV. This corresponds to 0.7 joules (J) absorbed per second by a 10-tzl sample. Fig. 2B shows a comparison between the absorbed flux at 2.8- and 2.54-GeV operation. At comparable beam currents, 2.8GeV operation yields about 1.4 times greater flux than 2.54-GeV operation.
Steady-State Hydroxyl Radical (. OH) Concentration For the range of X-ray energies produced at NSLS beam line X-9A, the interaction between the X rays and water is dominated by the photoelectric effect.2° In this interaction, the energy of an incoming photon is transferred to an electron, which is ejected from the water molecule. The ionized water molecule reacts with water to produce .OH, yielding 287 radicals for every 10 keV of energy thermalized in solution.2° The radicals can interact with a nucleic acid molecule or recombine to form H202 with a second-order rate constant of 5 × 109 M-is -1. As previously described, 17 the steady-state concentration of .OH can be estimated from the photon flux. A flux of 1014 photon/sec, as calculated for beam line X-9A operating at 2.54 GeV and 250 mA, corresponds to a dose rate of 1.6 × 10 -2 M/sec, which in turn yields a steady-state [.OH] of 1.2/zM.
Dose Response A parameter key to. the successful conduct of a synchrotron X-ray footprinting experiment is the amount of X-ray exposure ("dose") received by the RNA sample. Cleavage of the RNA must be controlled so that, on average, each molecule that is cleaved is cleaved only once. Exposures resulting in 10-30% RNA cleavage fulfill this requirement. 5 The correspondence between exposure to the X-ray beam and resulting cleavage of the nucleic acid is experimentally determined by a dose-response calibration. In these experiments, RNA labeled at one end with 32p is exposed to the X-ray beam for a series of exposure times at constant beam energy and current. Plotting the fraction of uncut Tetrahymena L-21 ScaI ribozyme as a function of time yields the dose-response c u r v e . 21 The fraction of uncut RNA is measured because this is conveniently assayed by quantitating the 20 N. V. Klassen, in, "Radiation Chemistry Principles and Applications," Austin, (I. Farhatazis and M. A. Rodgers, eds). VCH Publishers, Austin, Texas, 1987. 21 Dose-response curves obtained using the sample stand and electronic shutter (Fig. 1) or in the quench-flow apparatus (Fig. 4) under similar operating conditions are identical within experimental error. The dose-response curves shown were obtained using the sample stand and electronic shutter.
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decrease in density of the most slowly migrating band following denaturing polyacrylamide gel electrophoresis (PAGE) separation of the samples. 17 Figure 3A compares the dose-response relationships observed at 2.54-GeV (squares) and 2.8-GeV (circles) operation. The semilogarithmic relationship observed between RNA cleavage and X-ray exposure is predicted by Poisson statistics. 5 X-ray beam exposures of 10-25 and 3-9 msec for 2.54-
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Beam Current (mA) FIG. 3. (A) Dose-response curves for the L-21 ScaI R N A in 10 m M Mg 2+ u n d e r operating conditions of 2.54-GeV operation and 250 m A (squares) and 2.8-GeV operation and 240 m A (circles). T h e error in each d o s e - r e s p o n s e experiment was 13%. (B) Plot derived from several d o s e - r e s p o n s e curves at 2.54-GeV operation, showing exposure necessary for 30% cleavage u n d e r different operating conditions.
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and 2.8-GeV operation, respectively, are appropriate for footprinting the Tetrahymena ribozyme at the indicated ring current. Because the .OH cleavage statistics are dependent on the length of the RNA, a dose response is determined for each R N A studied by synchrotron x-ray footprinting. In addition, a calibration curve is required relating R N A cleavage to beam current at each of the two ring currents used at the NSLS since variations in the beam energy and current result in variable photon flux impacting samples. Figure 3B shows the experimentally determined X-ray exposures required to achieve 30% R N A cleavage of the Tetrahymena ribozyme as a function of beam current at 2.54 GeV. Because R N A cleavage and beam current are linearly related within the uncertainty of the measurements, a simple scaling factor is used to standardize beam exposure over the course of an experiment or series of experiments. Experimental Methods
Sample Storage At beam line X-9A all radiolabeled materials are stored within the experimental hutch. To protect the R N A from background X-ray exposure, both the R N A stock solutions and exposed samples are stored in acryliccovered lead-lined boxes (USA/Scientific Plastics, Ocala, FL). One lead box resides in a freezer that is within the experimental hutch (which in turn is covered by 1/16-in. lead sheathing) for sample storage. A second custom-ordered lead box that is 5 in. deep, resides on the worktable to allow easy access to samples. Samples are kept at 4° using cold-pack microfuge tube holders (USA/Scientific Plastics) or maintained at other temperatures using a temperature-regulated block placed within the lead box.
Electronic Shutterfor Manual Mixing Experiments For equilibrium studies and hand-mixing kinetic studies, an electronic shutter (Vincent Associates, Rochester, NY) impervious to the white light X-ray beam has replaced the "guillotine" gravity-driven shutter used previously. 15 The electronic X-ray shutter consists of a platinum/iridium alloy plate, 6.2 mm in diameter and 1 mm thick, capable of blocking X rays up to 30 keV in energy. A T-132 controller placed outside the hutch operates the shutter. Exposures ranging from 7 msec to 167 min can be programmed. The X-ray shutter is placed in front of a sample stand adjacent to the beam pipe (Fig. 1). Lead shielding minimizes X-ray scattering from the front of the shutter. The stand and the shutter are both aligned at the start of an
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experiment in order to ensure that the beam uniformly impacts the sample. The sample holder and shutter are mounted on slides so that they can be moved out of the beam path for rapid-mixing experiments. In typical equilibrium, dose-response or hand-mixing kinetics experiments, 10 /xl of sample is aliquoted into a microfuge tube and held by surface tension at the bottom. The tube is placed horizontally in a sample holder machined from a block of aluminum through which water from a temperature-regulated bath is circulated. Nominal temperature control of ___0.1° can be maintained with this apparatus. Following placement of the sample tube in the holder, the beam line enabling procedures are initiated and the safety shutter is opened, allowing the X-ray beam to enter the hutch. The electronic shutter is then triggered, followed by closure of the safety shutter and retrieval of the exposed sample.
Rapid-Mixing X-Ray Footprinting To conduct a rapid-mixing experiment, the X-ray shutter and sample holder are removed from the path of the beam. A steel "flight tube" is extended so that it is flush against the beam pipe at one end and against the modified Kin-Tek quench-flow apparatus (KinTek Corporation, Austin, Texas) on the other end (Fig. 1). In the Kin-Tek quench-flow apparatus, an exposure chamber 17aligned with the X-ray beam has replaced the sample loop selector (Fig. 4). Repositioning the exposure chamber to the back of the apparatus minimizes the air path of the beam by allowing the quench flow to be placed closer to the beam pipe. This arrangement maximizes the X-ray flux impacting the sample and minimizes the X-ray scattering that contributes to background degradation (see later discussion). The exposure chamber is now being machined from Vesbel, a material capable of withstanding prolonged exposure to the white light X-ray beam. The side of the cell facing the X-ray beam is covered with a thin Kapton window. The chamber dimensions are as previously describedJ 7 The sample flow train of the quench-flow device is siliconized (Surfa-Sil from Pierce Chemical Company, Rockford, IL) and treated with an anti-RNase agent (RNAse-ZAP from Ambion, Austin, TX) on a regular schedule to minimize the amount of absorbed RNA and sample degradation. The flight tube and lead shielding placed around the exposure chamber and on the face of the quench-flow apparatus minimize the scattered radiation generated as the X-ray beam passes through air (Fig. 1). The water surrounding the flow train of the apparatus additionally shields the RNA during an experiment. It is essential that a shield be placed over the syringe containing the [32p]RNA solution prior to enabling the beam line. A steel
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X-ra' ¢s FIG. 4. A schematic representation of the modified Kin-Tek quench-flow apparatus. Note that the entire sample flow train is immersed in the circulating water bath, ensuring efficient and uniform temperature equilibration.
tube (similar in thickness to the flight tube) mounted on a slide provides sufficient shielding yet is easily moved to provide access to the syringe. The time a sample is exposed to the X-ray beam is now determined by programming the rate at which the sample flows through the exposure chamber. This protocol more reliably exposes the entire RNA sample than the "push-pause" procedure previously described that it replaces. 17Sample exposure to the X-ray beam is dependent on the beam energy and current (as discussed earlier), length of the RNA, and the integrity of the RNA sample. Minimally background degraded [32p]RNA samples allow lower
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X-ray exposures to be used because sufficient experimental signal-to-noise ratios can be achieved with less cleavage.
Solutions TE buffer. 10 mM Tris-HC1, pH 7.5, 1 mM EDTA TEN buffer. 10 mM Tris-HC1, pH 7.5, 1 mM EDTA, 250 mM NaCI CE buffer. 10 mM sodium cacodylate, pH 7.5, 0.1 mM EDTA, pH 8.0 Mg2+ buffer. 10 mM sodium cacodylate, pH 7.5, 0.1 mM EDTA, pH 8.0, 20 mM MgC12 Precipitation buffer. 1.5 M sodium acetate, pH 5.0, 0.25 mg/ml tRNA in TE buffer 2× Gel loading buffer. 10 M urea, 0.2x TBE (0.089 M Tris-borate, 0.089 M boric acid, 2 mM EDTA, pH 8.0), 0.1% (w/v) bromphenol blue and xylene cyan.
Preparation of RNA RNA to be used in X-ray footprinting experiments is prepared by 5'end labeling of in vitro transcribed RNA with polynucleotide kinase as is generally used for biochemical analysis of RNA. 22 The RNA is prepared by T7 transcription from DNA templates followed by enzymatic removal of the 5'-triphosphate with calf alkaline intestinal phosphatase. The RNA is end labeled with [T-32p]ATP and polynucleotide kinase, followed by purification by PAGE. Gel slices containing the RNA are cut from the gel and soaked in TEN buffer at 4° overnight. The buffer is decanted to a collection tube and the gel slices washed with an additional 1.0 ml of TEN buffer. The combined TEN buffers are filtered and mixed with three volumes of absolute ethanol. The RNA is precipitated at -20 ° for 4-8 hr, pelleted by centrifugation, dissolved in 200 ~1 TE buffer and sequentially extracted with phenol followed by chloroform33 The precipitation is repeated a second time and the RNA dissolved in 30-50 tzl TE buffer and the specific radioactivity of the RNA determined by liquid scintillation counting. The [32P]RNA is brought to the beam line in 1-~Ci aliquots to a maximum of 10/~Ci pursuant to the dispersible radioactivity safety protocol in use at beam line X - 9 A . z4 In addition, aliquotting of the RNA reduces 22 A. J. Zaug, C. A. Grosshans, and T. R. Cech Biochemistry27, 8924 (1988). 23 T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982. 24 Copies of this safety protocol can be obtained from the Center for Synchrotron Biosciences. This protocol details the procedures and materials required for the transport and handling of dispersible radioactivity at the NSLS. Shipment of radioisotopes is subject to federal and state regulations.
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nonspecific degradation manifest as background in the gel autoradiograms. Typically, the 1-/zCi aliquots of the R N A are diluted to 20/xl using the buffer to be used in the planned experiments since prolonged storage of highly concentrated solutions of [32p]RNA often results in increased degradation and elevated background densities in the footprint autoradiograms. These aliquots of [32p]RNA are subsequently used to prepare the experimental samples, as described later.
Protocol for Manual Mixing X-Ray Footprinting Experiment The X-ray shutter is used for experiments that do not require rapid mixing of the R N A solution. Such experiments include determining doseresponse relationships, equilibrium titration experiments, and slow timescale kinetics experiments. The following example protocol is a slow timescale kinetics experiment for the Mg 2+ initiated folding of the Tetrahymena ribozyme. 1. An aliquot (typically 9/zl) of [32p]RNA diluted in assay buffer to an activity of -25-100,000 dpm is placed in a microfuge tube. One microliter magnesium buffer is added to the R N A solution to initiate the folding reaction yielding a final volume of 10/zl. For example, addition of 1 ttl of a 100 mM MgCI2 solution yields a final concentration of 10 mM Mg 2+. The solutions are mixed by a quick flick of the closed tube and the samples brought to the base of the tube if necessary by a brief centrifugation. The closed microfuge tube is placed in the sample stand. 2. The experimenter exits and interlocks the hutch. The beam line safety shutter can be opened after the 20-sec interlock safety pause. 3. After a time period several seconds shy of the desired reaction time, the safety shutter is opened allowing the X-ray beam to enter the hutch. The electronic X-ray shutter is immediately activated to expose the sample, thus minimizing exposure of the R N A to scattered radiation. The electronic shutter remains open for the programmed time as determined from doseresponse and calibration curves, as described earlier. The 20-sec interlock pause sets a minimum dead time for a manual experiment at - 3 0 sec. The exposure times typically range from 10 to 100 msec, depending on the length of the RNA. 4. The safety shutter is closed. The experimenter enters the hutch and retrieves the sample. 5. An equal volume of 2x loading buffer is added to the sample. Alternatively, precipitation buffer and three volumes of ethanol can be added to the sample. All samples are kept in a cold pack within a lead-fined box in the hutch both before and after exposure. Upon completion of the
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experiment, the samples are placed in the lead-lined freezer in the hutch until they are transferred to a laboratory for processing and analysis.
Protocol for Rapid-Mixing X-Ray Footprinting Experiment The protocol for conducting MgZ+-initiated quench-flow experiments has been modified from that previously describedS The [32p]RNA aliquots are stored in the lead-lined freezer in the hutch until ready for use. In addition to the experimental data points, control samples include R N A not exposed to the X-ray beam, R N A to which Mg 2+ is not added, and R N A to which Mg 2+ is added and allowed to equilibrate (i.e., a "fully folded" control). The latter two samples are exposed to the X-ray beam in the same manner as the experimental samples. 1. The circulating water bath is connected to the Kin-Tek apparatus, switched on, and allowed to equilibrate at the desired temperature of the experiment. 2. The drive syringes A, B, and C (Fig. 4) and the sample loop, reaction loop, and exposure chamber are rinsed successively with water and methanol and dried by pulling a vacuum through the sample flow train. 3. The first samples to be run are the "no-Mg 2+'' controls. For these samples, CE buffer is loaded into drive syringes A and C. Ethanol is loaded into middle syringe B. The valves are positioned so that the syringe is open only to the tubing below. The stepping motor is advanced until the plunger touches the syringes and the tubing between the syringes and the lower valves is filled with buffer. The mixing and reaction loops are sequentially rinsed with water and methanol and vacuum dried. 4. An aliquot of R N A (to a maximum specific radioactivity of 1/~Ci) is removed from the freezer, heated to 95° for 1 min, then diluted to a final volume of 200/.d in CE buffer. This volume of [3Zp]RNA is sufficient for ~15 samples. 5. A 1-ml sterile disposable syringe is filled with CE buffer and attached to inlet D. A second syringe is filled with the [3Zp]RNA solution and attached to inlet E (Fig. 4). 6. The solutions in D and E are pushed through the tubing up to marks that have been made on the sample loops indicating 10-/zl volumes. The sample valves are opened toward the drive syringes and the lead sleeve placed over the R N A containing syringe to protect it from scattered X-rays. 7. A microfuge tube containing 15/A precipitation buffer is attached to the exit line by a cap that has had a hole punched in it. The affixed tube is placed in a holder positioned under the quench flow. 8. The experimenter exits and interlocks the hutch. After 20 sec, the beam line safety shutter can be opened and the experiment initiated.
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9. Prior to allowing the X-ray beam to enter the hutch, the mixing time and exposure protocols are entered into a program downloaded to the motor controller. A "push-pause-push-push-push" timing sequence is used, slightly different from our original protocol. 17 The first "push" mixes the solutions driven by syringes D and E (the R N A solution and the Mg 2÷ buffer) by turbulent flow. The "pause" is the reaction time. The second "push" moves the mixed sample through the X-ray exposure chamber. The rate of fluid flow determines the duration of the X-ray exposure. The third "push" moves the sample to tip of the exit line, and the final "push" expels the sample into the collection tube. The final push is conducted at a slower speed in order to minimize splashing of the radioactive sample around the collection tube. The reaction time is the only variable changed over the course of a kinetics progress curve. 10. For reaction times <4 sec, the safety shutter is opened before the timing sequence is initiated. For reaction times >4 sec, the timing sequence is initiated and the safety shutter is opened just prior to the initiation of sample exposure, using a handheld timer as a guide. The safety shutter is closed immediately after the sample enters the collection tube. 11. The experimenter enters the hutch and retrieves the sample. The effluent from the quench flow consists of 200/xl of [32p]RNA and buffer and 100/zl ethanol. An additional 500/xl ethanol is added to the sample to initiate precipitation and the sample is stored in a cold pack within the lead-lined box in the hutch. 12. The mixing and reactions loops are rinsed successively with water and methanol and then vacuum dried in preparation for the next sample. 13. For the remainder of the samples in the Mg2+-initiated folding exper iment, the CE buffer in syringes A and D is removed and replaced with Mg 2+ buffer. 14. Steps 6 through 12 are repeated for the remainder of the samples. For each 15 samples, a new aliquot of RNA must be prepared as described in step 4. 15. The equilibrated, "fully folded" control samples provide evidence that the folding reaction has gone to completion. For these samples, an aliquot of R N A is diluted to 45/zl in CE buffer. Then 5 /zl of 100 mM Mg 2÷ buffer is added as a drop on the lid of the microfuge tube. The sample is heated to 95 ° for 1 min, then immediately spun down. This causes the Mg 2÷ to reach the solution when it is still warm, resulting in fast folding of the RNA. The fully folded control samples are loaded through inlet E and run as in steps 6 through 12. 16. On completion of the entire experiment, the samples are placed in the lead-lined freezer in the hutch until they are transferred to a laboratory for processing and analysis.
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Data Reduction and Analysis Analyses of the results of x-ray footprinting experiments are conducted as previously described in detail, t7 Briefly, the radiolysis reaction products are separated using PAGE. A storage phosphor screen and associated imager (Phosphorlmager, Molecular Dynamics, Sunnyvale, CA) are used to acquire a digital image of the electrophoretogram for densitometric analysis. Band intensity is quantitated using the Molecular Dynamics ImageQuant or equivalent image analysis software. The intensity within a protected region and a reference region is quantitated for each lane on the gel. The reference region is a set of bands that accounts for variability in sample loading in the individual lanes, but that shows the same degree of protection throughout the folding process. The densitometric results are exported to a spreadsheet (Microsoft Excel) for further processing and analysis. Each protected region is divided by the corresponding reference region in the same lane.
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Time (sec) FIG. 5. A representative kinetics progress curve d e t e r m i n e d for the protection that occurs at bases 118-120 of the Tetrahymena group I intron R N A . Fractional protection is plotted as a function of reaction time following addition of MgCI2 to a final concentration of 10 m M . T h e equilibrium folded control sample is shown by the open symbols plotted for convenience at 310 sec. T h e solid line depicts the best fit of the data to a single exponential with k = 0.2 _-_ 0.1 sec -1.
368
TECHNIQUES FOR MONITORING R N A CONFORMATION AND DYNAMICS
[23]
The resulting data points are plotted as a function of reaction time (for a kinetics experiment) or concentration (for an equilibrium experiment). A preliminary fit of the data to the appropriate model is conducted within Excel using the embedded program Magestic. Further analysis of progress or equilibrium curves can be accomplished using any number of nonlinear least-squares analysis programs and packages. Figure 5 shows a representative synchrotron X-ray footprinting progress curve obtained for the Mg2+-initiated folding of the Tetrahymenagroup I intron. The protection quantitated to obtain this curve represents bases 118-120 within the P4-P6 domain of the ribozyme and appears at a rate 0.2 sec -1. This protection is believed to represent a tertiary contact with bases within the peripheral domain P9.1 that show a comparable rate of protection. 15 In contrast, protections resulting from formation of the core of the P4-P6 domain occur at - 1 sec -1, those due to folding of the P5c domain at 2 sec 1, and formation of the catalytic core (domains P3-P7) at 0.03 sec -1.
Conclusion Synchrotron hydroxyl radical footprinting can be used to study folding of R N A on a millisecond timescale, and with single base resolution. Although this article focuses specifically on the application of synchrotron footprinting to R N A folding, a large number of systems are amenable to study by this technique. Studies of DNA-protein interactions, Mg 2+dependent D N A isomerization reactions, and protein folding are currently under way by the Center for Synchrotron Biosciences.
[231 A n a l y s i s o f G l o b a l C o n f o r m a t i o n o f B r a n c h e d R N A Species Using Electrophoresis and Fluorescence B y D A V I D M . J. LILLEY
Introduction Branch points are very common features of natural R N A molecules. These include asymmetrical bulges and helical junctions of various kinds, and an IUBMB nomenclature of these species is presented in Ref. 1. Natu1 D. M. J. Lilley, R. M. Clegg, S. Diekmann, N. C. Seeman, E. von Kitzing, and P. Hagerman, Eur. J. Biochern. 230, 1 (1995).
METHODS IN ENZYMOLOGY,VOL. 317
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