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[19] Following the folding of RNA with time-resolved synchrotron X-ray footprinting
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[19] F o l l o w i n g t h e F o l d i n g o f R N A w i t h T i m e - R e s o l v e d Synchrotron X-Ray Footprinting B y BIANCA SCLAVI, SARAH WOODSON, MICHAEL SULLIVAN, M A R K CHANCE, a n d MICHAEL BRENOWITZ
Introduction The conformations assumed by nucleic acids and conformational transitions play important roles in the molecular mechanisms by which nucleic acids perform their biological functions. As the study of nucleic acids enters its fifth decade following the proposal of the double helical structure of DNA, ~ the myriad pathways with which genetic information flows within organisms and the many roles played by nucleic acids in cellular metabolism continue to astonish. A clear example of the importance of conformational transitions is the ability of some RNA molecules (ribozymes) to fold into specific structures and attain catalytic activity.2 The Mg2+-dependent folding of the Tetrahymena thermophila L-21 Sca ribozyme is among the best characterized large RNA molecule with regard to its structure, folding, and enzymology? The fact that different domains of the ribozyme fold on time scales ranging from minutes to milliseconds4 makes it an ideal model system for the development of the synchrotron X-ray "footprinting" technique. Although this article focuses on the application of synchrotron X-ray footprinting to the " R N A folding problem," the principles and techniques that are presented are also readily applicable to problems of DNA and protein folding, DNA-protein, RNA-protein, and protein-protein interactions. A general discussion of applications of synchrotron X-ray footprinting being conducted by the Center for Synchrotron Biosciences has been published. 5
What Is Footprinting? Footprinting refers to assays that examine ligand binding and conformational changes by determning the solvent accessibility of the backbone of macromolecules through their sensitivity to chemical or enzymatic cleavage reactions. The key characteristics of a footprinting assay are that the cleavJ. D. Watson and F. H. C. Crick, Nature 171, 737 (1953). 2 A. M. Pyle and J. B. Green, Curt. Opin. Strucr BioL 5, 303 (1995). 3 Reviewed by T. R. Cech, "The RNA World" (R. F. Gesteland and J. F. Atkins, eds.), p. 239. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1993. 4 Reviewed by D. E. Draper, Nature Strucr Biol. 3, 397 (1996). 5 M. R. Chance, B. Sclavi, S. Woodson, and M. Brenowitz, Structure 5, 865 (1997).
METHODS IN ENZYMOLOGY,VOL.295
Copyright© 1998by AcademicPress All rightsof reproductionin any formreserved. 0076-6879/98 $25.00
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age of the backbone is limited, such that each position along the backbone is sampled with equal probability, and that the cleavage products are uniquely identified. For nucleic acids, the original implementation of footprinting used the endonuclease DNase I to study sequence-specific DNA-protein interactions. 6 Since that time, additional footprinting assays have been developed using a wide range of enzymatic and chemical nucleases. 7 Most pertinent to this article are footprinting assays developed using the hydroxyl radical (.OH), which is commonly generated in biochemistry and molecular biological laboratories by the Fenton-Haber-Weiss reaction using FeEDTA as a catalyst. 8
Quantitative Footprinting Quantitative protocols have been developed for the conduct of DNase I footprinting experiments for the determination of thermodynamic 9 and kinetic 1°,al constants describing protein-DNA interactions. I2 These protocols have been successfully extended to three different implementations of -OH footprinting. ~3-15 The key advantage of quantitative footprinting techniques is their ability to monitor transitions with single base resolution for nucleic acids. Individual-site binding isotherms ~6 and individual-site kinetic progress curves n determined from quantitative thermodynamic and kinetic footprinting studies, respectively, provide an ensemble of local mea-
6 D. Galas and A. Schmitz, Nucleic Acids Rev. 5, 3157 (1978); A. Schmitz and D. Galas, Nucleic Acids Res. 6, 111 (1979), 7 A. Revzin, ed., "Footprinting Techniques for Studying Nucleic Acid-Protein Complexes." Academic Press, New York, 1993. 8 T. D. Tullius, B. A. Dombroski, M. E. Churchill, and L. Kam, Methods Enzymol. 155, 537 (1987). 9 M. Brenowitz, D. F. Senear, M. A. Shea, and G. K. Ackers, Methods Enzymol. 130, 132 (1986); M. Brenowitz, D. F. Senear, M. A. Shea, and G. K. Ackers, Proc. Nat. Acad. Sci. U.S.A. 83, 8462 (1986); K. S. Koblan, D. L. Bain, D. Beckett, M. A. Shea, and G. K. Ackers, Methods Enzymol. 210, 405 (1982); D. F. Senear and D. W. Bolen, Methods Enzymol. 210, 463 (1992). 10V. Petri, M. Hsieh, and M. Brenowitz, Biochemistrty 34, 9977 (1995). ll M. Hsieh and M. Brenowitz, Methods EnzymoL 274, 478 (1996). 12 V. Petri and M. Brenowitz, Curr. Opin. Biotechnol. 8~ 36 (1997). i3 p. A. King, V. E. Anderson, J. O. Edwards, G. Gustafson, R. C. Plumb, and J. W. Suggs, Z Am. Chem. Soc. 114, 5430 (1992); P. A. King, E. Jamison, D. Strahs, V. E. Anderson, and M. Brenowitz, Nucleic Acids Res. 21, 2473 (1993). 14 D. Strahs and M. Brenowitz, J. Mol. Biol. 244, 494 (1994). is B. Sclavi, S. Woodson, M. Sullivan, M. Chance, and M. Brenowitz, J. Mol. Biol. 266, 144 (1997). 16 G. K. Ackers, A. D. Johnson, and M. A. Shea, Proc. Natl. Acad. Sci. U.S.A. 79, 1129 (1982); G. K. Ackers, M. A. Shea, and F. R. Smith, J. Mol. Biol. 170, 223 (1983).
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sures of global macromolecular transitions from which detailed energetic and mechanistic "portraits" can be painted. A quench-flow DNase ! footprinting technique 11 has achieved time scales on the order of tens of milliseconds for kinetic measurements using rapid mixing technology. However, it is unlikely that this technique can be extended to shorter time scales due to the exceptionally high nuclease concentrations that are required to achieve them. The same limitation holds true for solution conditions, such as high salt concentrations, unfavorable to DNA binding by DNase I. In addition, the sizable Stokes radius of most nucleases limits their sensitivity to structural changes that occur within protein binding sites, as the probe is physically excluded from all the nucleotides within a footprint. Thus, clear temporal and structural limitations to the extension of this technique to other areas of inquiry exist. •O H F o o t p r i n t i n g
•OH has proven to be a valuable probe of nucleic acid structure and nucleic acid-protein interactions because it provides structural resolution with single base pair resolution8,17 and is equally applicable to DNA and RNA. 15 In addition to Fe-EDTA, peroxonitrous acid 13,~8and radiolysis~5'19'2° have been used successfully to conduct .OH footprinting studies• Quantitative •OH footprinting has been applied successfully to problems of both proteinDNA interactions and RNA folding/3,14,15,21Several studies strongly suggest that nucleic acid cleavage by .OH is predominantly dependent on the solvent accessibility of the phosphodiester b a c k b o n e 22-24 and is relatively insensitive to base sequence and whether the nucleic acid is single or double stranded. 21 Changes in RNA tertiary but not secondary structure can be detected by .OH footprinting. However, •OH reactivities not readily interpretable as changes in solvent accessibility are also observed. The small 17 T. D. Tullius and B. A. Dombroski, Proc. NatL Acad. Sci. U.S.A. 83, 5469 (1986). 18 M. Gotte, R. Marquet, C. Isel, V. E. Anderson, G. Keith, H. J. Gross, C. Ehresmann, B. Ehresmann, and H. Heumann, FEBS Lett. 390(2), 226 (1996). 19 j. j. Hayes, L. Kam, and T. D. Tullius, Methods Enzymol. 186, 545 (1990); M. A. Price and T. D. Tullius, Methods Enzymol. 212, 194 (1992). 2o j. Franchet-Beuzit, M. Spotheim-Maurizot, R. Sabattier, B. Blazy-Gaudras, and M. Charlier, Biochemistry 32, 2104 (1993). 21 D. C. Celander and T. R. Ceeh, Biochemistry 29, 1355 (1990); D. C. Celander and T. R. Cech, Science 251, 401 (1991). 22 W. J. Dixon, J. J. Hayes, J. R. Levin, M. F. Weidner, B. A. Dombroski, and T. D. Tullius, Methods EnzymoL 208, 380 (1991). 23 j. A. Latham and T. R. Cech, Science 245, 276 (1989). 24 j. H. Cate, A. R. Gooding, E. Podell, K. Zhou, B. L. Golden, C. E. Kundrot, T. R. Cech, and J. A. Doudna, Science 273, 1678 (1996).
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Stokes radius of .OH results in sensitivity to local changes in the conformation of unprotected positions within protein binding sites. 1437
Cleavage Time Scales of Different Methods of .OH Footprinting An advantage of Fe-EDTA mediated -OH footprinting is that inexpensive and commonly available reagents are utilized. However, Fe-EDTA mediated cleavage of D N A and R N A on time scales less than tens of seconds has not been obtainable. 15 An alternative chemistry for conducting a .OH footprinting probe uses peroxonitrous acid. t3 An advantage of this reagent is that stable solutions of peroxonitrite can be readily prepared and used in the laboratory; reactions are initiated by simply adding the reagent to the sample solution. However, the accessible time scale is limited by the ~2-sec half-life of peroxonitrite disproportionation. In addition, the pH dependence of the disproportionation reaction limits the applicability of this reagent to neutral pH. Radiolytic footprinting using low flux, high energy sources requires exposures of tens of minutes to hours. ~9'2°
Synchrotron X-ray Footprinting Synchrotron X-ray footprinting is a melding of radiolytic chemistry using a high flux X-ray source with stopped-flow mixing technology; this technique permits the examination of nucleic acid dynamics with single nucleotide structural resolution, sensitivity to solvent accessibility, parsimonious use of the nucleic acid substrate, and millisecond time resolution, s'ls Radiolysis generates .OH according to Eq. (1)25: H20
H20 ~ H20 + + easy ~ H30 + + .OH + eaq
(1)
Photons interact with water (as described in more detail later) and generate a water ion and an electron. The water ion reacts with another water molecule within a few picoseconds, generating .OH and a hydronium ion. Meanwhile, the electron becomes "hydrated." Irradiation of aqueous solutions with various forms of radiation (such as y rays,/3 particles, or fast neutrons) result in basically the same ionizations of water. However, irradiation from sources with a low total flux requires exposure times on the order of hours in order to conduct footprinting experiments. In contrast, an unfocused "white beam" bending magnet beamline at the National Synchrotron Light Source (NSLS) delivers -1014-1015 photons, sec -1 of a continuous spectrum of X-rays with an effective energy range of 3-30 keV in 2s N. V. Klassen, in "Radiation Chemistry Principles & Applications" (Farhatazis and M. A. Rodgers, eds.), p. 29. VCH, Texas, 1987.
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a beam characterized by relatively little divergence. The flux provided by NSLS beamline X-19C results in DNA and RNA footprinting with beamline exposures of tens of milliseconds.15Beamline exposures as short as 10 msec are now possible for nucleic acid footprinting experiments on the newly commissioned beamline X-9A of the Center for Synchrotron Biosciences (Fig. 1). Unlike nuclease- or chemical reagent-based footprinting techniques, this shortening of the footprinting time scale to as little as tens of milliseconds is obtained without the need to add high concentrations of chemical reagents to the solution of macromolecules. This technique differs from those familiar to most molecular biologists and biochemists in that a synchrotron is expensive and immovable and time on a beamline is limited and precious. Therefore, careful planning is required to conduct these X-ray footprinting experiments. Although our laboratories now benefit from access to a dedicated beamline at the NSLS, X-9A, the development of this technique was accomplished through "General User" time that is available on a peer-reviewed proposal basis at most synchrotrons. It is possible for investigators interested in using this technique to gain access to a beamline by such a mechanism. The following section of this protocol is intended to provide investigators with the basic information required to conduct X-ray footprinting experiments as general users. Of course, the specifications and configuration will aid those individu-
1 0.9 0.8 0.7
0 0.6 C 0.5
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0
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0.3
0.2 0
20
40
60
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X-ray exposure (reset)
FIG. 1. D o s e - r e s p o n s e curve relating the amount of R N A that remains uncleaved to the time of X-9A beam exposure as described in the text. The top band on the electrophoresis gels, representing the full-length nucleic acid molecules, was quantitated. The shaded region indicates the time of X-ray beam exposure required to nick 10-30% of the nucleic acids as required for conducting footprinting experiments.
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als fortunate enough to be in the position to establish a footprinting beamline of their own to accomplish this goal. Synchrotron X-ray footprinting provides improved time resolution compared to other time-resolved footprinting techniques with high structural resolution and general applicability to the study of DNA, RNA, and proteins. The remainder of this article presents the protocols that have been developed for the conduct of synchrotron X-ray footprinting experiments to monitor the folding of the Tetrahymena thermophila L-21 Sca ribozyme. These protocols provide the foundation for X-ray footprinting studies and will be generally applicable to other macromolecules. Because a decision to utilize synchrotron technology represents a major commitment of time and resources, the nature of the experimental problem being addressed and the applicability of this technology to the problem must be carefully considered before experimental studies are initiated. The initial portion of the experimental methods section is a discussion of radiolytic chemistry and synchrotron technology. The intent of this initial discussion is to provide investigators with an understanding of the strengths and limitations of this approach and to foster the implementation of this approach at other synchrotrons. Because detailed protocols and discussions of the issues related to the conduct and analysis of quantitative footprinting experiments have been published for thermodynamic, 9'26 quench-flow kinetic, n and .OH footprinting 14'15 studies, general issues common to these techniques will only be briefly touched on in this article. Experimental Methods Radiolysis and •OH Chemistry
The radiolysis of water is a well-understood process and can be accomplished using a wide variety of radiation sources. Most of the knowledge in this area has been derived from pulse radiolysis studies, and a number of standard texts in the area can be consulted, z7 Interactions of high-energy photons or particles with matter involve primarily pair-production, Compton effects, and the photoelectric effect, where the relative contribution of the three processes is correlated with the particle or photon energy, 26 M. Brenowitz and D. F. Senear, in "Current Protocols in Molecular Biology" (F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl, eds.), Wiley, New York, 1989; M. Brenowitz, D. F. Senear, E. Jamison, and D. D. DalmaWeiszhausz, in "Footprinting Techniques for Studying Nucleic Acid-Protein Complexes" (A. Revzin, ed.), p. 1. Academic Press, New York, 1993. 27 Farhatazis and M. A. Rodgers, eds., "Radiation Chemistry Principles and Applications." VCH, Texas, 1987.
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respectively. Most of the energy ultimately absorbed tends to eject electrons from the atoms of the material. These processes are primarily dependent on the atomic composition of the material, unlike interactions of infrared, visible, or ultraviolet light, which are primarily dependent on the molecular structure. This provides a fundamental distinction between the interactions of ionizing and nonionizing radiation with matter. In the case of X-rays in the keV energy range interacting with water, the photoelectric effect and Compton scattering are primarily involved. The former effect dominates X-ray/water interactions at and below energies of 20 keV. The absorption of the photon promotes an electron to an unbound state with kinetic energy equal to the incident photon energy minus the ionization energy (Compton scattering also results in the production of secondary electrons). These electrons and the water ions are responsible for all subsequent radiation chemistry in dilute aqueous solutions. The electrons are thermalized and deposit their energy in discrete ionizations of other water molecules. The ionized water molecules react with water to produce .OH according to the reaction outlined in Eq. (1). It is important to note that all effects of radiation chemistry (for ionizing radiation) in dilute aqueous solutions involve direct interactions only with water (at - 5 5 M concentration) whereas the radiation effects on the solutes are entirely indirect. This can be contrasted with radiolysis of cells or tissue, where both direct and indirect effects are important to the chemistry. The initial interactions of ionizing radiation with water occur in "spurs" localized around the radiation-absorbing water molecules. The chemistry within the spur involves the reactants on the right-hand side of Eq. (1) and is complete within 100 nsec, yielding the following major products 25 4.14 H20 l(~v 2.7e~q + 2.7 H ÷ + 0.61 H. + 2.87 "OH + 0.43 H 2 + 0.61 H 2 0 2 + 0.03 H O 2
(2)
For every 100 eV of energy absorbed, 2.87 .OH are produced (i.e., for each 10 keV photon absorbed followed by thermalization of the high-energy electrons produced, 287 .OH molecules are produced). The reactions within the spurs are inhomogeneous with respect to bulk solution, whereas the products on the right-hand side of Eq. (2) are homogeneous, e.g., these species are free to react with radical scavengers or macromolecules following second-order kinetics. The stoichiometric coefficients on the right-hand side of the equation are called G values. These G values can be converted to standard units by multiplying by 0.104. Thus, the yield of .OH is 0.3 /xmol J 1 of absorbed energy.2s 28 H. Winick, in "Synchrotron Radiation Sources" (A. Primer and H. Winick, eds.), p. 1. World Scientific, Singapore, 1994.
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Nucleic Acid Cleavage Using Synchrotron X-Rays Based on these G values, the photon flux and energy distribution of a synchrotron radiation source, and the absorption coefficient of X-rays by water, the [.OH] generated for various synchrotron configurations can be predicted. For bending magnet and wiggler sources, the spectrum of radiation is a smooth continuum defined by a single parameter called the critical energy, or ec, which is given in Eq. (3) 28 ec = 0.665 BE 2
(3)
where ec is in units of keV, E is in units of GeV, and B is in units of tesla. Half the power is radiated below this energy and half above. The NSLS storage ring circulates relativistic electrons with energies (E) of 2.584 GeV with a current maximum beam current of 350 mA, while the bending magnets operate at a magnetic field (B) of 1.25 tesla. For the X-9A bending magnet beamline, the critical energy is 5.6 keV. The electrons circulate in discrete bunches or packets with durations of 0.6 nsec. The overall pulse structure at NSLS includes 25 such bunches of 0.6 nsec duration for every ring orbit, with an orbit period of 556 nsec. Thus, the synchrotron dose is a quasicontinuous wave. As will become clear later, the electron bunch structure must be taken into consideration in designing experiments. As the beam interacts with water in the first few hundreds of microseconds, the [.OH] builds up as energy is thermalized according to Eq. (2). In addition, the radicals also recombine based on second-order kinetics, E q . (4)29: k2
•OH + .OH ~ H202,
k2 = 5 X 10 9 M - l s e c -1
(4)
Thus the influence of two competing processes must be considered when calculating the effects of the radiation. First we will consider the dose of radiation. For beamline X-9A, approximately 10 9 photons/sec/mm2/0.07% bandwidth are available at 350 mA beam current (approximately 15 m from the source). For a sample with a l-ram path length (and 10 #1 volume), considering the energy distribution of the white beam, the absorption coefficient of water, and operating at 350 mA, approximately 1017 e g is thermalized per second. Using the G value for .OH, the dose rate, D, is - 5 × 10 -4 M" sec -a. The dose can also be estimated calorimetrically. The X-9A beam causes -0.3 ° per second rise in the temperature of the irradiated solution (at 230 mA). Because 4.1 J causes a 1° rise in a solution of 1 ml of water, and the yield of .OH is 0.3 t~mol J 1 of absorbed energy, we 29 A. Hummel, in "Radiation Chemistry Principles and Applications (Farhatazis and M. A. Rodgers, eds.), p. 97. VCH, Texas, 1987.
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estimate a dose rate of -4.5 x 10 -4 M sec -1 (at 350 mA ring current) by this method. This estimate is a lower limit due to imperfect insulation. The rate of loss of -OH is shown in Eq. (5)29: -d['OHI dt
- D - 2 k2[-OH] 2
(5)
The factor of 2 on the right-hand side adjusts for the stochiometry, e.g., one .OH is made in the radiolysis while two are lost for each bimolecular recombination. At the steady-state concentration, the dose rate equals the loss due to bimolecular recombination. For a dose rate, D, of 5 × 10 -4 M:sec -1, with k2 = 5 X 109 M 1 s e c - l , [ ' O H ] steady state = 2 × 10 -7 M. As the intensity of beam is increased, the [.OH] goes up, as does bimolecular recombination. However, if the dose is increased by x, the steady state concentration goes up by V~x. The "cutting power" of the radiation source equals the steady-state [.OH] times the duration of exposure (actually the integrated concentration of radicals times the duration of exposure). This analysis is comparable to that developed for Cot (concentration times time) analysis of DNA hybridization of short (<400 bp) fragments of DNA. 3° The footprinting time scale for beamline X-9A is - 2 0 msec, thus the Cot value for footprinting at an [.OH] steady state of 2 x 10 -7 M is 4 × 10 -9 M.sec. This value should be taken into consideration when evaluating synchrotron and radiation sources for footprinting. The need for quenching the radicals may not be necessary based on the particular experiment to be performed, as self-quenching occurs rapidly. For example, the lifetime for the .OH produced is shown in Eq. (6) 29 1 tl/2 - ['OH]- k2
(6)
where [-OH] is the initial concentration of .OH (2 x 10-7 M when the beam is turned off) and k2 is 5 × 10 9 M -t sec -1. Thus, the tl/2 is 1 msec when the beam exposure ceases. The result of the dependence of tl/2 on the initial concentration is that once the initial concentration has been reduced to one-half, the lifetime is then twice as long. Therefore we can calculate the expected additional cutting that occurs after beam exposure ceases until the reaction is quenched, either by stopped flow injection of quenching solution or, in a hand-mixing experiment, until hand quenching is performed. For example, for the first milliseconds after the sample moves out of the beam, the concentration averages 1.5 × 10 -7 M with a Cot value of 1.5 x 10-1° M.sec. This indicates that 1% additional cutting occurs in 30 R. J. Britten and D. E. Kohne, "Repeated Segments of D N A " (Offprint 1173) Scientific American, April and references cited therein, 1970.
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this first millisecond. An identical Cot value can be calculated for each additional tl/2. For the folding experiments performed in this paper, where quenching occurs 15-20 msec after the beam exposure ceases, a maximum of 2.0-3.0% additional cleavage occurs while the sample flows from the exposure chamber into the receiving tube and is diluted or quenched. Given this, calculation of the cleavage characteristics of synchrotron sources before experiments commence is straightforward. However, a brief comparison of the synchrotron to other radiation sources is instructive. For example, Van de Graft accelerators have been used extensively for pulse radiolysis and can easily provide [.OH] of 5-25 ~M in centimeter path length cells for kinetics studies. Are these sources suitable for time-resolved X-ray footprinting? The tl/2 for an initial, single dose of 5 ~M is 40/~sec, giving a Cot value of 1.5 × 10-l° M.sec. Thus, less than 4% of the Cot required for footprinting accrues in the first tin. Because the accumulated dose for each tl/2 is equal, over 25 ta/2 values must elapse to accumulate an equivalent dose. Because the tl/2 value doubles after every hi2, it takes 160 msec to achieve 50% of the Cot value of the synchrotron dose. In fact, even if initial [.OH] of several mM could be generated instantaneously, nucleic acid cleavage equivalent to beamline X-9A could not be achieved in 20 msec. The discussion now returns to the electron bunch structure of the synchrotron and its quasicontinuous wave nature. The synchrotron delivers a constant and reproducible 200 nM [.OH]. Reproducible nucleic acid cleavage is essential to quantitative footprinting. In considering a source for radiolysis, the repetition rate should be kHz or higher to assure a sufficient and reproducible dose. Other disadvantages of the Van de Graft (and many other "single shot" sources) are the high energy of the source (3 MeV for the Brookhaven Van de Graft), requiring the use of longer path lengths (cm versus ram) and thus more biological material (ml versus txl). Also, the "peak to peak" intensity of the device varies considerably, making it difficult to assure an equivalent dose for each time point of a kinetic determination.
Millisecond Exposure Shutter An X-ray shutter capable of millisecond exposures is an important tool for calibrating the exposure of samples to the beamline and for conducting equilibrium experiments. A schematic of the "guillotine shutter" that we designed and use is shown in Fig. 2. The shutter consists of a ¼in. steel plate with an adjustable slit (maximum length 100 ram) that can be dropped along a track by releasing a solenoid from outside the experimental hutch.
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FIG. 2. Schematic representation of the X-ray shutter used for manually exposing samples to the X-ray beam.
When the steel plate is at rest the sample is protected from the X-ray beam. Because the gravitational acceleration is constant, X-ray exposure is readily controlled by the length of the slit. A gas piston can be added to the shutter in order to damp the descent of the plate and extend the range of exposures available. The advantages of this design are simplicity and reliability. Details of the shutter design may be provided on request.
Rapid Mixing X-Ray FootprintingDevice A modified Kin-Tek three-syringe quench-flow device 31 is being used to rapidly mix R N A and Mg 2+ and then exposure the sample mixture to the X-ray beam for the appropriate length of time (Fig. 3). An apparatus of the same type is being used routinely for quench-flow DNase I kinetics studies of protein-DNA interactions, la Although the Kin-Tek apparatus has three drive syringes, only two are absolutely required for X-ray footprinting experiments. However, as will be shown later, the third syringe can be used to mix a radical scavenger with the nucleic acid samples immediately following exposure to the X-ray beam. The other basic requirement for a suitable apparatus is a drive motor whose function and speed can be precisely controlled from a remote location. The modifications required to 31 j. Langowski, C. Urbanke, and E. Schuppe, Anal Biochem. 142, 91 (1984); K. A. Johnson, Methods Enzymol. 134, 677 (1986).
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SteppingMotor I Controller
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adapt the Kin-Tek apparatus for X-ray work were the replacement of the sample loop selector with an X-ray exposure chamber and reprogramming of the control software (see later). Placement of a lead shield on the front of the mixer surrounding the exposure chamber minimizes damage to the Plexiglas box surrounding the mixing components. An additional lead shield is used to protect the R N A sample present in its loading syringe from
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scattered radiation (Fig. 3). Siliconizing the flow path of the mixer minimizes absorption of R N A to the walls of its tubes. 32 Several features of this apparatus have demonstrated advantages for the execution of X-ray footprinting experiments that should be looked for in adapting other rapid mixing devices for X-ray beamline use. First, the drive syringes, plumbing, and exposure chamber are all enclosed in a circulating water bath. In addition to providing temperature control, the water bath provides an effective shield against scattered radiation within the experimental hutch. Second, the apparatus can be controlled remotely from outside the experimental hutch by simply making an extension for the cable linking the stepping motor and its controller. Third, modification of the apparatus to accept the X-ray exposure chamber was readily accomplished. Fourth, customization of the control software is straightforward. The major modification to the mixing device required for X-ray footprinting experiments was the insertion of an X-ray transparent "exposure chamber" following the "reaction loop" in the solution flow patch (Fig. 3). A schematic drawing of the exposure chamber is shown in Fig. 4. The 25-/xl chamber is machined out of Lexan with the front window made of X-ray transparent Kapton. The l-ram thickness of the flow cell ensures complete penetration of the sample solution by the X-ray beam. The fittings and tubing required to install the chamber were purchased from the Lee Company (Westbrook, CT). Ten microliters of each of the reactants is loaded into separate 3.5-cm lengths of 0.8-mm internal diameter tubing ("sample loops"). The sample loops intersect at a "T junction" with the 6-cm-long reaction loop that is off sufficient volume to hold the combined volume of the reactants (25/zl). The proximal end of the reaction loop connects with the exposure chamber. A final length of tubing is connected to the distal end of the exposure chamber and exits the apparatus. A second T junction can be placed in the flow train after the exposure chamber should the use of the third syringe be desired to mix the radiolyzed samples with a radical quench solution. The modifications that were made to the mixer required reprogramming of the software controlling the stepping motor in order to conduct the 32 Absorption of nucleic acids to the walls of the tubes has only been observed for sample reaction times greater than 2 rain. The acquisition of time points on the order of minutes is not required for progress curves with millisecond time scales. However, because parts of the Tetrahymena ribozyme fold with time scales on the order of minutes, the acquisition of slow folding data with the rapid mixer is desirable in order to assure the consistency of the fast and slow folding progress curves. Unpublished data have established the consistency of manual and rapid mixing methods for reactions such as the slow folding of the core of the Tetrahymena ribozyme.
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small screws
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FIG. 4. The X-ray exposure chamber. The chamber is machined out of Lexan. All surfaces contacting the sample are rounded so as to maintain smooth solution flow and to minimize sample loss.
X-ray footprinting protocol that is described later. The modifications to the software provided by KinTek were coded and downloaded from the personal c o m p u t e r being used to control the mixer-using utilities supplied by the manufacturer. The modified control software may be provided on request.
Calibrating Volumes of Rapid Mixing Device The general procedures for calibrating the flow rates and volumes of the Kin T e k mixer are provided in the documentation provided by the m a n u f a c t u r e r and have been published. 11 Briefly, the sample loops volumes are determined using a solution containing a known amount of radiolabeled material. One of the sample loops is filled with the radiolabeled solution, the valve is then turned so that it is open toward the syringe, and about 250/xl is expelled by pushing on the syringe. By quantitating the amount of radioactive material in the expelled volume, the volume of each of the sample loops, the loading loops, and the mixing loop can be determined. In addition, the amount of solution that is expelled from the rapid mixer following one revolution of the m o t o r (=5000 steps) should be determined at the m o t o r speed of the experiment. This is to calibrate the n u m b e r of steps necessary to push the sample into the reaction loop, up to the exposure chamber, and then to fill the exposure chamber. The position of the solution meniscus can be confirmed visually during the calibration procedure.
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Fro. 5. Schematic of setup in the experimental hutch.
Setting up Beamline for X-Ray Footprinting The simplest and most flexible placement of the equipment in the experimental hutch is to position the rapid mixer behind the manual shutter as close to the front port of the beamline as possible (Fig. 5). This arrangement allows both manual and rapid mixing experiments to be interchangeably conducted simply by locking the shutter in the open position for rapid mixing experiments. Of course, both apparatuses need to be aligned with the X-ray beam. Placing the samples close to the front port of the beamline minimizes the X-ray exposure times by minimizing scattering of the beam by a i r y The control wires for both devices are run out of the hutch through standard ports to the experimental control station. The small size of the equipment facilitates setup and breakdown of the instruments, allowing efficient use of limited periods of beam time.
Achieving Appropriate Cleavage of Nucleic Acids by .OH A number of factors affect the amount of cleavage of a nucleic acid sample and must be controlled and calibrated. The appropriate amount of 33Beam scattering can also be minimizedby the insertion of an argon-filledor vacuum "flight tube" in the path of the beamline in situations where the presence of other equipment precludes placing the shutter and mixer close to the front port.
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nucleic acid cleavage is 10-30%, at which level, on average, each molecule that is cleaved by .OH is cleaved only once. In order to achieve this level of cleavage, a dose-response calibration should be completed under the reaction conditions of the experiment. The following principal factors affect X-ray cleavage of nucleic acids. 1. The flux of X-rays impacting on a sample varies among different beamlines and as a function of the operative beamline parameters. Important characteristics include the amount (mrads) of X-ray radiation incident on the sample. This is partially determined by the proximity of the hutch to the storage ring and/or the presence of focusing optics (no focusing is present on beamline X-9A). The beamline path, including beryllium and or aluminum windows, as well as the distance in air that the X-ray beam travels within the beamline hutch, must be considered (for X-9A, a 0.5-mm Be window and a 100-/xm AI window are present, as well as a 1.5-m air path). The flux of the beam also depends on the "ring current" of a synchrotron ring, which decreases from its maximum level following injection of the storage ring (currently 350 mA at NSLS) because of energy loss by the electrons encountering gas molecules in the ring vacuum. The ring current may decrease by as much as 50% between "fills." Calibration experiments must be performed to determine the appropriate dose for various values of the ring current. 2. Solution conditions that affect nucleic acid cleavage include temperature, pH, and the chemical composition of the buffer. Commonly used buffers such as Tris and HEPES are effective radical scavengers. Their use will dramatically increase the X-ray exposure required to footprint. 15 Phosphate, cacodylate, citrate, and pyrophosphate buffers are good alternative buffers. Glycerol, present in many biological reagent storage buffers, is also a potent radical scavenger; as little as 0.1% (v/v) will inhibit nucleic acid cleavage by 50%. Other radical scavengers include urea and ethanol.
Preparation of RNA and Reagents The standard precautions for handling RNA-containing solutions are followed during all of the steps of the X-ray footprinting protocol. RNasefree reagents and deionized (18 M r ) water are used in the preparation of all R N A samples. Buffers are placed on ice immediately following thawing of stock solutions. The Tetrahymena thermophila L-21 ScaI ribozyme is transcribed with T7 R N A polymerase from plasmid pT7L-21 digested with
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ScaI and purified by Sephadex G-50 chromatography. 34 The R N A is 5' labeled with [y-32p]ATP and polynucleotide kinase and then purified by electrophoresis on a 4% denaturing acrylamide gel following published protocols. 3s The 32p-labeled R N A can be stored in TE buffer at either 4 ° or - 2 0 °. Required Solutions TE buffer: 10 mM Tris-HC1, pH 7.5, 1 mM EDTA Assay buffer: 10 mM sodium cacodylate, pH 7.5; 0.1 mM EDTA, pH 8.0 Magnesium buffer: 10 mM sodium cacodylate, pH 7.5; 0.1 mM EDTA, pH 8.0; 20 mM MgCI2 Magnesium buffer for folded samples: 10 mM sodium cacodylate, pH 7.5; 0.1 mM EDTA, pH 8.0; 10 mM MgCI2 Precipitation solution: 1.5 M sodium acetate, pH 5.0; 0.25 mg/ml tRNA (in TE buffer) TBE buffer: 0.089 M Tris-base, 0.089 M boric acid, 0.002 M EDTA (from 0.5 M EDTA stock solution at pH 8.0) 2× gel-loading buffer: 10 M Urea, 0.2× TBE, 0.1% (w/v) bromphenol blue and 0.1% (w/v) Xylene Cyanol.
Protocol for Manual Mixing X-Ray Footprinting Experiment This manual mixing protocol is used for all experiments that require rapid detection. These experiments include calibration of acid cleavage by X-rays from a beamline, equilibrium titration ments, and kinetics experiments with time scales on the order nutes.
do not nucleic experiof mi-
1. The R N A and Mg 2+ solutions (previously equilibrated at the desired temperature) are mixed by adding 2/zl of 50 mM magnesium buffer to 8/xl of the R N A solution (-30,000 cpm). The tube is then placed sideways in a sample holder 36 behind the shutter. The sample is held to the bottom of the microfuge tube by surface tension. In order to ensure that the entire sample is exposed to the X-ray beam, the surface area of the sample must be smaller in diameter than the beam and the depth of the sample should not exceed 1 mm (although 34 A. J. Zaug, C. A. Grosshans, and T. R. Cech, Biochemistry 27, 8924 (1988). 35 j. A. Latham, A. J. Zaug, and T. R. Cech, Methods Enzymol. 181, 558 (1990). 36 The sample holder can be a temperature-regulated block in order to conduct experiments at temperatures other than ambient.
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this is energy dependent). A 10-t~l sample volume is optimal in the current configuration of NSLS beamline X-9A. 2. The safety procedures are initiated that allow entry of the X-ray beam into the vacated and locked beamline hutch. These procedures require abut 30 sec for completion. 3. The safety shutter of the beamline hutch is enabled, allowing entry of the X-ray beam. A second or two later, the steel plate of the shutter is allowed to drop, exposing the sample to the X-ray beam. The safety shutter is closed immediately following the conclusion of the experiment, turning off the X-ray beam. The operator enters the hutch to retrieve the sample. This procedure minimizes exposure of the nucleic acid sample to scattered X-ray radiation. 4. An equal volume of 2× loading buffer or precipitation buffer and three volumes of absolute ethanol are added to the sample. Samples are stored on ice outside the beamline hutch until the entire experiment is completed before further processing.
Protocol for Rapid Mixing X-Ray Footprinting Experiment The experimental protocol described here was adapted from that developed for quench-flow DNase I footprinting11; the rationale and background that are common to the X-ray and DNase I kinetic footprinting techniques will not be repeated here. A schematic of the rapid mixing device is shown in Fig. 3. An overview of a MgZ+-initiated quench-flow kinetics RNA folding experiment is illustrated in Fig. 6 and is described later. This protocol can be readily modified to accommodate other folding initiators and to conduct protein-nucleic acid binding studies. 1. The circulating water bath for the rapid mixture is turned on and the apparatus is allowed to equilibrate at the desired temperature. Fifty microliters of the assay buffer is placed in the water bath to equilibrate to temperature. The flow train of the mixer is washed sequentially with deionized water and methanol and dried with a vacuum. 2. The drive syringes A and C are loaded with the assay buffer to be used in the experiments. The valves are positioned so that the syringe is open toward the outside feed; once the syringe is filled (with no air bubbles) the valves are moved so that the syringe is open toward the tubing beneath it but not toward the outside. (Syringe B can be loaded with a solution such as 10 mM thiourea to be mixed with the sample immediately on exiting the exposure chamber. An alternative procedure is to place an aliquot of a quench
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PUSH
397 PUSH
7 F1G.6. Schematicrepresentation of the sequence of events required to obtain each experimental data point in a synchrotron X-ray footprinting kinetics experiment.
solution in the collection tube. 37) The apparatus is then primed with the assay buffer by moving the motor so that it touches both syringes and the buffer from the syringes has filled all the tubing. 3. One-milliliter disposable syringes (C and D in Fig. 3) are loaded with the R N A sample in assay buffer or magnesium buffer. The magnesium buffer is used to initiate the R N A folding reaction. The assay buffer-containing syringe is used for the control samples in which the unfolded R N A is footprinted. Both syringes remain attached to the outside of the Plexiglas box housing the mixer. The mixer is rinsed and dried as in step 1. 4. The appropriate volumes of the RNA-containing solution and magnesium buffer are pushed into the left and right sample loops, respectively, up to the marks indicating 10/xl (5 mm from the valve). (For the "unfolded control" sample, the R N A is mixed with Mg2+-free 37The -OH cleavage reaction may need to be quenched depending on the time scale of the transition, see earlier. Control experimentsfor the RNA foldingreaction conducted with and without a thiourea quench solution in syringe B were indistinguishable (unpublished data).
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assay buffer.) The syringe containing the RNA is covered with a 5.
6. 7.
8.
9. 10.
lead sleeve to protect it from scattered X-ray radiation. The valves are then turned so that they are open toward the drive syringes. The exit tube is inserted through a microfuge top that has a small hole punched in it. A microfuge tube containing 15/zl precipitation buffer is secured to the end of the exit tube. At this point the investigator exits the beamline hutch and initiates the safety procedures required for enabling the beamline. The timing sequence for the sample is entered into the drive motor controller at this time to inject and mix the samples and subsequently expose them to the X-ray beam. Each injection consists of a "pushpause-push-pause-push-push" timing sequence. The first push mixes the samples. The first pause is the time the reagents are allowed to react. This is the only timing parameter that is changed during an experiment; it is usually incremented to cover the course of the expected duration of the folding reaction. The second push moves the sample into the X-ray exposure chamber. The second pause determines the exposure to the X-ray beam. (The exposure to the X-ray beam is equal to the pause time, plus the time it takes the sample to enter and exit the exposure chamber, about 5 msec at the 180-rpm motor speed utilized). The third push moves the sample out of the exposure chamber. The final push is conducted at a slower velocity in order to minimize splashing of the sample as it is expelled into the microfuge collection tube. The safety shutter of the beamline hutch is enabled, allowing entry of the X-ray beam. The timing sequence for the mixer is initiated and a data point is collected. The safety shutter is closed immediately following the conclusion of the experiment (turning off the X-ray beam), and the operator enters the hutch to retrieve the RNA sample. For samples that react for greater than 10 sec, the timing sequence is initiated prior to enabling the X-ray beam in order to minimize the time that the X-rays are entering the experimental hutch. The controller software has been programmed with a countdown timer that allows the experimenter to know when the sample is poised to enter the exposure chamber. Three volumes (300/xl) of cold ethanol are added to the irradiated sample in order to precipitate the RNA. The mixer is washed and dried as described in step 1. The next collection tube is secured to the exit tube. Step 2 is conducted as necessary. Steps 4-9 are repeated until the desired progress curve has been obtained, as described later.
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Mge+-Initiated RNA-Folding Experiment The following protocol describes in detail the conduct of an experiment to follow the kinetics of the Mg>-dependent folding of the Tetrahymena L-21 ScaI ribozyme. Because beam time may be limited, it is essential that the integrity of the biological reagents be checked prior to conducting a synchrotron X-ray footprinting experiment.
Sample and Reagent Preparation 11. Prepare 20-30 ml of the assay buffer, which will be used to fill the drive syringes (5 ml each) and for MgZ+-free samples that do not require magnesium. 12. Precipitation buffer (15/zl) is added to the desired number of siliconized 1.5-ml microfuge tubes. Twenty-eight samples are prepared for a typical folding experiment. 13. Approximately 1.5 × 106 cpm of the [32p]RNA to be used in the experiment is taken from a stock solution (stored at 4°), heated for 1 min at 90°, and then placed in a 42° water bath for 15 min. Thirty to fifty thousand cpm of the [32p]RNA is used for each reaction. The [32p]RNA is diluted in assay buffer to a total volume of 480 /zl. An equal amount of the magnesium buffer is also prepared at this time. These volumes are in excess of what is required for a 28sample experiment to allow for unforeseen events and for loss of sample during the cleaning procedure. 14. Folded R N A (120/zl) is prepared by adding 1.2/xl of 1 M MgC12 to an aliquot of the sample solution prepared in the preceding step. This control sample is incubated at 42 ° for the duration of the experiment (see step 18). A solution of 10 mM MgC12 (120/~1) is also prepared at this time. 15. Place approximately 30 ml of absolute ethanol on ice. 16. Steps 1-3 are conducted to set up the rapid mixer. 17. The first sample to be footprinted is an unfolded control. Steps 3-10 are conducted for two R N A samples using Mg2+-free assay buffer. Following completion of these samples, the right-hand sample port is washed and dried, a syringe containing magnesium buffer is secured to the inlet port, and the new buffer is loaded into the sample loop. 18. The next samples to be footprinted define the RNA-folding transition. Steps 4-10 are again repetitively conducted using magnesium buffer, incrementing the reaction time for each sample. All other timing parameters remain constant throughout the experiment (see step 7).
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19. Because footprinting defines a transition from an initial to a final state, which is converted to "fractional protection" by numerical methods (described in the next section), it is essential that a significant number of data points be acquired for both the initial and the final portions of the kinetics progress curve. A typical kinetics experiment spans several decades in time scale. 20. The last sample to be footprinted is a folded RNA sample. The RNA and magnesium buffer syringes are removed, and the loops are rinsed and dried as described earlier. The RNA sample prepared in step 14 is mixed with the 10 mM Mg 2+ solution. This sample provides confirmation that the folding reaction has gone to completion. 21. The RNA samples from a complete kinetics footprinting experiment (or a series of experiments) that have been kept on ice outside the beamline hutch are brought back to the laboratory and processed for polyacrylamide gel electrophoresis. 9'1l'15 Dried polyacrylamide gels are exposed to phosphor storage screens and scanned. A Phosphorlmager (Molecular Dynamics, Inc., Sunnyvale, CA) is used in our laboratories for this purpose. Alternative devices are commercially available.
Analysis of X-Ray Footprinting Experiments The intensities of the electrophoretic bands present on the digital autoradiograms are analyzed using the Image Quant (Molecular Dynamics) software provided with our scanner? 8 Although the density changes of individual bands can be quantitated separately, improved precision is obtained by quantitating all of the bands within a "block. ''39 The bands within a block are boxed using the tools available in the software. Another block is defined within each lane as a standard; the standard is a band or bands whose density does not change during the folding reaction. When the blocks of interest have been defined for an experiment, the integrated density of each block is calculated and the results exported to a Microsoft Excel spreadsheet. 4° The integrated density of each folding "block" is divided by 38 A number of commercially available programs can be utilized to analyze the images obtained from the gel electrophoretograms. The most important criterion is the ability to process the full 16-bit density resolution of the scanned images. Detailed descriptions of this analysis procedure have been published. 9 39 m "block" is defined as a group of bands whose densities change equivalentlY as either a function or ligand or time for equilibrium or kinetics experiments, respectivelyY 4o The ImageQuant software is integrated with the Microsoft spreadsheet. Other analysis programs can be substituted through the implementation of the appropriate export and conversion functions.
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that of the standard in the lane in order to normalize for variations in the total density of each lane due to sample loss during precipitation or gel loading. Block density data are paired with the reaction time appropriate for the lane to generate the kinetic progress curves. These operations are facilitated through the application of "macros" within the spreadsheet. This densitometric analysis of the digital image(s) of the electrophoretogram of a kinetic "footprint" experiment yields P i , the "apparent protection." Y, the fractional protection of the binding site, is obtained by fitting data to the coupled equations P = Plower + (Pupper -- Plower) Y
(7a)
Y
(7b)
= 1 -
e TM
where p is the "apparent saturation," P~ow~rand P,pper are the lower and upper limits of the transition curve, ka is the first-order association rate constant, and t is time. The initial analyses of these curves are fit using the Magestic fitting program (Logix Consulting, Inc., www.lgx.com) as a macro within Excel and are scaled to Y using the best-fit values of Pupper and P~..... Further analysis of the progress curves, including global analysis of multiple curves, is conducted using the program NONLIN as has been described. 9'~1 The 65% confidence limits estimated in this manner correspond to approximately one standard deviation. When multiple data sets are analyzed globally, each data set is weighted by the inverse of the square root of the variance of its individual fit. Extensive discussions of the issues that must be considered in conducting and analyzing quantitative nuclease protection experiments have been published. 9
1.2
Io • •
1.0
8
•
•
o---J
•
0.8
•
J
•
]
~'o.~ 0.4 0.2
0.0 0
10
20
30 60
Seconds
FIG. 7. Millisecond RNA folding progress curve of bases 212-215 in the P4 helix in the
Tetrahymena thermophila group I intron. The progress curve is composed of four independent experiments analyzed globally. The open symbols represent the "folded control" in which an RNA sample was preequilibrated with Mg2+ prior to injection into the rapid mixer.
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Representative Result for Mg2+-DependentFolding of Tetrahymena Ribozyme An example of a kinetics progress curve obtained on a millisecond timescale is shown in Fig. 7 for the protection of bases 212-215 of the P4-P6 domain of the Tetrahymenaribozyme. The ability of the synchrotron X-ray footprinting progress curves to resolve subsecond rate constants can be seen readily in this example; experimental data have been obtained for virtually all of the reaction. The progress curve is described by a single exponential within the precision of the experiment. The folding rate of this site is among the fastest measured within this ribozyme (0.9 sec-1), and its protection is due to the interaction of the A bulge in P5a with the P4 helix, one of the first steps in the folding pathway. 41 The protection of the Abulge bases from .OH occurs at the same rate, whereas the protection of other bases, e.g., those that make contact with the P9 domain, occur at a slower rate (0.4 see-l). Summary The rapid mixing synchrotron X-ray footprinting technique described in this article allows nucleic acid folding and ligand binding reactions to be followed on a millisecond time resolution with single nucleotide resolution. In principle, the change in .OH protection of every nucleotide in a nucleic acid hundreds of nucleotides long can be monitored separately, In addition, a wide range of solution conditions are compatible with the radiolytic generation of -OH. These characteristics of synchrotron X-ray footprinting create opportunities for conducting thermodynamic and kinetic studies of nucleic acids that are both comprehensive and detailed. Kinetic footprinting studies of a number of systems have been initiated by the Center for Synchrotron Biosciences using this technique.
41 B. Sclavi, M. Sullivan, M. R. Chance, M. Brenowitz, and S. A. Woodson, Science 279, 1940 (1998).
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[19] F o l l o w i n g t h e F o l d i n g o f R N A w i t h T i m e - R e s o l v e d Synchrotron X-Ray Footprinting B y BIANCA SCLAVI, SARAH WOODSON, MICHAEL SULLIVAN, M A R K CHANCE, a n d MICHAEL BRENOWITZ
Introduction The conformations assumed by nucleic acids and conformational transitions play important roles in the molecular mechanisms by which nucleic acids perform their biological functions. As the study of nucleic acids enters its fifth decade following the proposal of the double helical structure of DNA, ~ the myriad pathways with which genetic information flows within organisms and the many roles played by nucleic acids in cellular metabolism continue to astonish. A clear example of the importance of conformational transitions is the ability of some RNA molecules (ribozymes) to fold into specific structures and attain catalytic activity.2 The Mg2+-dependent folding of the Tetrahymena thermophila L-21 Sca ribozyme is among the best characterized large RNA molecule with regard to its structure, folding, and enzymology? The fact that different domains of the ribozyme fold on time scales ranging from minutes to milliseconds4 makes it an ideal model system for the development of the synchrotron X-ray "footprinting" technique. Although this article focuses on the application of synchrotron X-ray footprinting to the " R N A folding problem," the principles and techniques that are presented are also readily applicable to problems of DNA and protein folding, DNA-protein, RNA-protein, and protein-protein interactions. A general discussion of applications of synchrotron X-ray footprinting being conducted by the Center for Synchrotron Biosciences has been published. 5
What Is Footprinting? Footprinting refers to assays that examine ligand binding and conformational changes by determning the solvent accessibility of the backbone of macromolecules through their sensitivity to chemical or enzymatic cleavage reactions. The key characteristics of a footprinting assay are that the cleavJ. D. Watson and F. H. C. Crick, Nature 171, 737 (1953). 2 A. M. Pyle and J. B. Green, Curt. Opin. Strucr BioL 5, 303 (1995). 3 Reviewed by T. R. Cech, "The RNA World" (R. F. Gesteland and J. F. Atkins, eds.), p. 239. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1993. 4 Reviewed by D. E. Draper, Nature Strucr Biol. 3, 397 (1996). 5 M. R. Chance, B. Sclavi, S. Woodson, and M. Brenowitz, Structure 5, 865 (1997).
METHODS IN ENZYMOLOGY,VOL.295
Copyright© 1998by AcademicPress All rightsof reproductionin any formreserved. 0076-6879/98 $25.00
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age of the backbone is limited, such that each position along the backbone is sampled with equal probability, and that the cleavage products are uniquely identified. For nucleic acids, the original implementation of footprinting used the endonuclease DNase I to study sequence-specific DNA-protein interactions. 6 Since that time, additional footprinting assays have been developed using a wide range of enzymatic and chemical nucleases. 7 Most pertinent to this article are footprinting assays developed using the hydroxyl radical (.OH), which is commonly generated in biochemistry and molecular biological laboratories by the Fenton-Haber-Weiss reaction using FeEDTA as a catalyst. 8
Quantitative Footprinting Quantitative protocols have been developed for the conduct of DNase I footprinting experiments for the determination of thermodynamic 9 and kinetic 1°,al constants describing protein-DNA interactions. I2 These protocols have been successfully extended to three different implementations of -OH footprinting. ~3-15 The key advantage of quantitative footprinting techniques is their ability to monitor transitions with single base resolution for nucleic acids. Individual-site binding isotherms ~6 and individual-site kinetic progress curves n determined from quantitative thermodynamic and kinetic footprinting studies, respectively, provide an ensemble of local mea-
6 D. Galas and A. Schmitz, Nucleic Acids Rev. 5, 3157 (1978); A. Schmitz and D. Galas, Nucleic Acids Res. 6, 111 (1979), 7 A. Revzin, ed., "Footprinting Techniques for Studying Nucleic Acid-Protein Complexes." Academic Press, New York, 1993. 8 T. D. Tullius, B. A. Dombroski, M. E. Churchill, and L. Kam, Methods Enzymol. 155, 537 (1987). 9 M. Brenowitz, D. F. Senear, M. A. Shea, and G. K. Ackers, Methods Enzymol. 130, 132 (1986); M. Brenowitz, D. F. Senear, M. A. Shea, and G. K. Ackers, Proc. Nat. Acad. Sci. U.S.A. 83, 8462 (1986); K. S. Koblan, D. L. Bain, D. Beckett, M. A. Shea, and G. K. Ackers, Methods Enzymol. 210, 405 (1982); D. F. Senear and D. W. Bolen, Methods Enzymol. 210, 463 (1992). 10V. Petri, M. Hsieh, and M. Brenowitz, Biochemistrty 34, 9977 (1995). ll M. Hsieh and M. Brenowitz, Methods EnzymoL 274, 478 (1996). 12 V. Petri and M. Brenowitz, Curr. Opin. Biotechnol. 8~ 36 (1997). i3 p. A. King, V. E. Anderson, J. O. Edwards, G. Gustafson, R. C. Plumb, and J. W. Suggs, Z Am. Chem. Soc. 114, 5430 (1992); P. A. King, E. Jamison, D. Strahs, V. E. Anderson, and M. Brenowitz, Nucleic Acids Res. 21, 2473 (1993). 14 D. Strahs and M. Brenowitz, J. Mol. Biol. 244, 494 (1994). is B. Sclavi, S. Woodson, M. Sullivan, M. Chance, and M. Brenowitz, J. Mol. Biol. 266, 144 (1997). 16 G. K. Ackers, A. D. Johnson, and M. A. Shea, Proc. Natl. Acad. Sci. U.S.A. 79, 1129 (1982); G. K. Ackers, M. A. Shea, and F. R. Smith, J. Mol. Biol. 170, 223 (1983).
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sures of global macromolecular transitions from which detailed energetic and mechanistic "portraits" can be painted. A quench-flow DNase ! footprinting technique 11 has achieved time scales on the order of tens of milliseconds for kinetic measurements using rapid mixing technology. However, it is unlikely that this technique can be extended to shorter time scales due to the exceptionally high nuclease concentrations that are required to achieve them. The same limitation holds true for solution conditions, such as high salt concentrations, unfavorable to DNA binding by DNase I. In addition, the sizable Stokes radius of most nucleases limits their sensitivity to structural changes that occur within protein binding sites, as the probe is physically excluded from all the nucleotides within a footprint. Thus, clear temporal and structural limitations to the extension of this technique to other areas of inquiry exist. •O H F o o t p r i n t i n g
•OH has proven to be a valuable probe of nucleic acid structure and nucleic acid-protein interactions because it provides structural resolution with single base pair resolution8,17 and is equally applicable to DNA and RNA. 15 In addition to Fe-EDTA, peroxonitrous acid 13,~8and radiolysis~5'19'2° have been used successfully to conduct .OH footprinting studies• Quantitative •OH footprinting has been applied successfully to problems of both proteinDNA interactions and RNA folding/3,14,15,21Several studies strongly suggest that nucleic acid cleavage by .OH is predominantly dependent on the solvent accessibility of the phosphodiester b a c k b o n e 22-24 and is relatively insensitive to base sequence and whether the nucleic acid is single or double stranded. 21 Changes in RNA tertiary but not secondary structure can be detected by .OH footprinting. However, •OH reactivities not readily interpretable as changes in solvent accessibility are also observed. The small 17 T. D. Tullius and B. A. Dombroski, Proc. NatL Acad. Sci. U.S.A. 83, 5469 (1986). 18 M. Gotte, R. Marquet, C. Isel, V. E. Anderson, G. Keith, H. J. Gross, C. Ehresmann, B. Ehresmann, and H. Heumann, FEBS Lett. 390(2), 226 (1996). 19 j. j. Hayes, L. Kam, and T. D. Tullius, Methods Enzymol. 186, 545 (1990); M. A. Price and T. D. Tullius, Methods Enzymol. 212, 194 (1992). 2o j. Franchet-Beuzit, M. Spotheim-Maurizot, R. Sabattier, B. Blazy-Gaudras, and M. Charlier, Biochemistry 32, 2104 (1993). 21 D. C. Celander and T. R. Ceeh, Biochemistry 29, 1355 (1990); D. C. Celander and T. R. Cech, Science 251, 401 (1991). 22 W. J. Dixon, J. J. Hayes, J. R. Levin, M. F. Weidner, B. A. Dombroski, and T. D. Tullius, Methods EnzymoL 208, 380 (1991). 23 j. A. Latham and T. R. Cech, Science 245, 276 (1989). 24 j. H. Cate, A. R. Gooding, E. Podell, K. Zhou, B. L. Golden, C. E. Kundrot, T. R. Cech, and J. A. Doudna, Science 273, 1678 (1996).
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Stokes radius of .OH results in sensitivity to local changes in the conformation of unprotected positions within protein binding sites. 1437
Cleavage Time Scales of Different Methods of .OH Footprinting An advantage of Fe-EDTA mediated -OH footprinting is that inexpensive and commonly available reagents are utilized. However, Fe-EDTA mediated cleavage of D N A and R N A on time scales less than tens of seconds has not been obtainable. 15 An alternative chemistry for conducting a .OH footprinting probe uses peroxonitrous acid. t3 An advantage of this reagent is that stable solutions of peroxonitrite can be readily prepared and used in the laboratory; reactions are initiated by simply adding the reagent to the sample solution. However, the accessible time scale is limited by the ~2-sec half-life of peroxonitrite disproportionation. In addition, the pH dependence of the disproportionation reaction limits the applicability of this reagent to neutral pH. Radiolytic footprinting using low flux, high energy sources requires exposures of tens of minutes to hours. ~9'2°
Synchrotron X-ray Footprinting Synchrotron X-ray footprinting is a melding of radiolytic chemistry using a high flux X-ray source with stopped-flow mixing technology; this technique permits the examination of nucleic acid dynamics with single nucleotide structural resolution, sensitivity to solvent accessibility, parsimonious use of the nucleic acid substrate, and millisecond time resolution, s'ls Radiolysis generates .OH according to Eq. (1)25: H20
H20 ~ H20 + + easy ~ H30 + + .OH + eaq
(1)
Photons interact with water (as described in more detail later) and generate a water ion and an electron. The water ion reacts with another water molecule within a few picoseconds, generating .OH and a hydronium ion. Meanwhile, the electron becomes "hydrated." Irradiation of aqueous solutions with various forms of radiation (such as y rays,/3 particles, or fast neutrons) result in basically the same ionizations of water. However, irradiation from sources with a low total flux requires exposure times on the order of hours in order to conduct footprinting experiments. In contrast, an unfocused "white beam" bending magnet beamline at the National Synchrotron Light Source (NSLS) delivers -1014-1015 photons, sec -1 of a continuous spectrum of X-rays with an effective energy range of 3-30 keV in 2s N. V. Klassen, in "Radiation Chemistry Principles & Applications" (Farhatazis and M. A. Rodgers, eds.), p. 29. VCH, Texas, 1987.
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a beam characterized by relatively little divergence. The flux provided by NSLS beamline X-19C results in DNA and RNA footprinting with beamline exposures of tens of milliseconds.15Beamline exposures as short as 10 msec are now possible for nucleic acid footprinting experiments on the newly commissioned beamline X-9A of the Center for Synchrotron Biosciences (Fig. 1). Unlike nuclease- or chemical reagent-based footprinting techniques, this shortening of the footprinting time scale to as little as tens of milliseconds is obtained without the need to add high concentrations of chemical reagents to the solution of macromolecules. This technique differs from those familiar to most molecular biologists and biochemists in that a synchrotron is expensive and immovable and time on a beamline is limited and precious. Therefore, careful planning is required to conduct these X-ray footprinting experiments. Although our laboratories now benefit from access to a dedicated beamline at the NSLS, X-9A, the development of this technique was accomplished through "General User" time that is available on a peer-reviewed proposal basis at most synchrotrons. It is possible for investigators interested in using this technique to gain access to a beamline by such a mechanism. The following section of this protocol is intended to provide investigators with the basic information required to conduct X-ray footprinting experiments as general users. Of course, the specifications and configuration will aid those individu-
1 0.9 0.8 0.7
0 0.6 C 0.5
Z C
0.4
0
I,L
0.3
0.2 0
20
40
60
80
X-ray exposure (reset)
FIG. 1. D o s e - r e s p o n s e curve relating the amount of R N A that remains uncleaved to the time of X-9A beam exposure as described in the text. The top band on the electrophoresis gels, representing the full-length nucleic acid molecules, was quantitated. The shaded region indicates the time of X-ray beam exposure required to nick 10-30% of the nucleic acids as required for conducting footprinting experiments.
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als fortunate enough to be in the position to establish a footprinting beamline of their own to accomplish this goal. Synchrotron X-ray footprinting provides improved time resolution compared to other time-resolved footprinting techniques with high structural resolution and general applicability to the study of DNA, RNA, and proteins. The remainder of this article presents the protocols that have been developed for the conduct of synchrotron X-ray footprinting experiments to monitor the folding of the Tetrahymena thermophila L-21 Sca ribozyme. These protocols provide the foundation for X-ray footprinting studies and will be generally applicable to other macromolecules. Because a decision to utilize synchrotron technology represents a major commitment of time and resources, the nature of the experimental problem being addressed and the applicability of this technology to the problem must be carefully considered before experimental studies are initiated. The initial portion of the experimental methods section is a discussion of radiolytic chemistry and synchrotron technology. The intent of this initial discussion is to provide investigators with an understanding of the strengths and limitations of this approach and to foster the implementation of this approach at other synchrotrons. Because detailed protocols and discussions of the issues related to the conduct and analysis of quantitative footprinting experiments have been published for thermodynamic, 9'26 quench-flow kinetic, n and .OH footprinting 14'15 studies, general issues common to these techniques will only be briefly touched on in this article. Experimental Methods Radiolysis and •OH Chemistry
The radiolysis of water is a well-understood process and can be accomplished using a wide variety of radiation sources. Most of the knowledge in this area has been derived from pulse radiolysis studies, and a number of standard texts in the area can be consulted, z7 Interactions of high-energy photons or particles with matter involve primarily pair-production, Compton effects, and the photoelectric effect, where the relative contribution of the three processes is correlated with the particle or photon energy, 26 M. Brenowitz and D. F. Senear, in "Current Protocols in Molecular Biology" (F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl, eds.), Wiley, New York, 1989; M. Brenowitz, D. F. Senear, E. Jamison, and D. D. DalmaWeiszhausz, in "Footprinting Techniques for Studying Nucleic Acid-Protein Complexes" (A. Revzin, ed.), p. 1. Academic Press, New York, 1993. 27 Farhatazis and M. A. Rodgers, eds., "Radiation Chemistry Principles and Applications." VCH, Texas, 1987.
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respectively. Most of the energy ultimately absorbed tends to eject electrons from the atoms of the material. These processes are primarily dependent on the atomic composition of the material, unlike interactions of infrared, visible, or ultraviolet light, which are primarily dependent on the molecular structure. This provides a fundamental distinction between the interactions of ionizing and nonionizing radiation with matter. In the case of X-rays in the keV energy range interacting with water, the photoelectric effect and Compton scattering are primarily involved. The former effect dominates X-ray/water interactions at and below energies of 20 keV. The absorption of the photon promotes an electron to an unbound state with kinetic energy equal to the incident photon energy minus the ionization energy (Compton scattering also results in the production of secondary electrons). These electrons and the water ions are responsible for all subsequent radiation chemistry in dilute aqueous solutions. The electrons are thermalized and deposit their energy in discrete ionizations of other water molecules. The ionized water molecules react with water to produce .OH according to the reaction outlined in Eq. (1). It is important to note that all effects of radiation chemistry (for ionizing radiation) in dilute aqueous solutions involve direct interactions only with water (at - 5 5 M concentration) whereas the radiation effects on the solutes are entirely indirect. This can be contrasted with radiolysis of cells or tissue, where both direct and indirect effects are important to the chemistry. The initial interactions of ionizing radiation with water occur in "spurs" localized around the radiation-absorbing water molecules. The chemistry within the spur involves the reactants on the right-hand side of Eq. (1) and is complete within 100 nsec, yielding the following major products 25 4.14 H20 l(~v 2.7e~q + 2.7 H ÷ + 0.61 H. + 2.87 "OH + 0.43 H 2 + 0.61 H 2 0 2 + 0.03 H O 2
(2)
For every 100 eV of energy absorbed, 2.87 .OH are produced (i.e., for each 10 keV photon absorbed followed by thermalization of the high-energy electrons produced, 287 .OH molecules are produced). The reactions within the spurs are inhomogeneous with respect to bulk solution, whereas the products on the right-hand side of Eq. (2) are homogeneous, e.g., these species are free to react with radical scavengers or macromolecules following second-order kinetics. The stoichiometric coefficients on the right-hand side of the equation are called G values. These G values can be converted to standard units by multiplying by 0.104. Thus, the yield of .OH is 0.3 /xmol J 1 of absorbed energy.2s 28 H. Winick, in "Synchrotron Radiation Sources" (A. Primer and H. Winick, eds.), p. 1. World Scientific, Singapore, 1994.
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Nucleic Acid Cleavage Using Synchrotron X-Rays Based on these G values, the photon flux and energy distribution of a synchrotron radiation source, and the absorption coefficient of X-rays by water, the [.OH] generated for various synchrotron configurations can be predicted. For bending magnet and wiggler sources, the spectrum of radiation is a smooth continuum defined by a single parameter called the critical energy, or ec, which is given in Eq. (3) 28 ec = 0.665 BE 2
(3)
where ec is in units of keV, E is in units of GeV, and B is in units of tesla. Half the power is radiated below this energy and half above. The NSLS storage ring circulates relativistic electrons with energies (E) of 2.584 GeV with a current maximum beam current of 350 mA, while the bending magnets operate at a magnetic field (B) of 1.25 tesla. For the X-9A bending magnet beamline, the critical energy is 5.6 keV. The electrons circulate in discrete bunches or packets with durations of 0.6 nsec. The overall pulse structure at NSLS includes 25 such bunches of 0.6 nsec duration for every ring orbit, with an orbit period of 556 nsec. Thus, the synchrotron dose is a quasicontinuous wave. As will become clear later, the electron bunch structure must be taken into consideration in designing experiments. As the beam interacts with water in the first few hundreds of microseconds, the [.OH] builds up as energy is thermalized according to Eq. (2). In addition, the radicals also recombine based on second-order kinetics, E q . (4)29: k2
•OH + .OH ~ H202,
k2 = 5 X 10 9 M - l s e c -1
(4)
Thus the influence of two competing processes must be considered when calculating the effects of the radiation. First we will consider the dose of radiation. For beamline X-9A, approximately 10 9 photons/sec/mm2/0.07% bandwidth are available at 350 mA beam current (approximately 15 m from the source). For a sample with a l-ram path length (and 10 #1 volume), considering the energy distribution of the white beam, the absorption coefficient of water, and operating at 350 mA, approximately 1017 e g is thermalized per second. Using the G value for .OH, the dose rate, D, is - 5 × 10 -4 M" sec -a. The dose can also be estimated calorimetrically. The X-9A beam causes -0.3 ° per second rise in the temperature of the irradiated solution (at 230 mA). Because 4.1 J causes a 1° rise in a solution of 1 ml of water, and the yield of .OH is 0.3 t~mol J 1 of absorbed energy, we 29 A. Hummel, in "Radiation Chemistry Principles and Applications (Farhatazis and M. A. Rodgers, eds.), p. 97. VCH, Texas, 1987.
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SYNCHROTRON X-RAY FOOTPRINTING
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estimate a dose rate of -4.5 x 10 -4 M sec -1 (at 350 mA ring current) by this method. This estimate is a lower limit due to imperfect insulation. The rate of loss of -OH is shown in Eq. (5)29: -d['OHI dt
- D - 2 k2[-OH] 2
(5)
The factor of 2 on the right-hand side adjusts for the stochiometry, e.g., one .OH is made in the radiolysis while two are lost for each bimolecular recombination. At the steady-state concentration, the dose rate equals the loss due to bimolecular recombination. For a dose rate, D, of 5 × 10 -4 M:sec -1, with k2 = 5 X 109 M 1 s e c - l , [ ' O H ] steady state = 2 × 10 -7 M. As the intensity of beam is increased, the [.OH] goes up, as does bimolecular recombination. However, if the dose is increased by x, the steady state concentration goes up by V~x. The "cutting power" of the radiation source equals the steady-state [.OH] times the duration of exposure (actually the integrated concentration of radicals times the duration of exposure). This analysis is comparable to that developed for Cot (concentration times time) analysis of DNA hybridization of short (<400 bp) fragments of DNA. 3° The footprinting time scale for beamline X-9A is - 2 0 msec, thus the Cot value for footprinting at an [.OH] steady state of 2 x 10 -7 M is 4 × 10 -9 M.sec. This value should be taken into consideration when evaluating synchrotron and radiation sources for footprinting. The need for quenching the radicals may not be necessary based on the particular experiment to be performed, as self-quenching occurs rapidly. For example, the lifetime for the .OH produced is shown in Eq. (6) 29 1 tl/2 - ['OH]- k2
(6)
where [-OH] is the initial concentration of .OH (2 x 10-7 M when the beam is turned off) and k2 is 5 × 10 9 M -t sec -1. Thus, the tl/2 is 1 msec when the beam exposure ceases. The result of the dependence of tl/2 on the initial concentration is that once the initial concentration has been reduced to one-half, the lifetime is then twice as long. Therefore we can calculate the expected additional cutting that occurs after beam exposure ceases until the reaction is quenched, either by stopped flow injection of quenching solution or, in a hand-mixing experiment, until hand quenching is performed. For example, for the first milliseconds after the sample moves out of the beam, the concentration averages 1.5 × 10 -7 M with a Cot value of 1.5 x 10-1° M.sec. This indicates that 1% additional cutting occurs in 30 R. J. Britten and D. E. Kohne, "Repeated Segments of D N A " (Offprint 1173) Scientific American, April and references cited therein, 1970.
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this first millisecond. An identical Cot value can be calculated for each additional tl/2. For the folding experiments performed in this paper, where quenching occurs 15-20 msec after the beam exposure ceases, a maximum of 2.0-3.0% additional cleavage occurs while the sample flows from the exposure chamber into the receiving tube and is diluted or quenched. Given this, calculation of the cleavage characteristics of synchrotron sources before experiments commence is straightforward. However, a brief comparison of the synchrotron to other radiation sources is instructive. For example, Van de Graft accelerators have been used extensively for pulse radiolysis and can easily provide [.OH] of 5-25 ~M in centimeter path length cells for kinetics studies. Are these sources suitable for time-resolved X-ray footprinting? The tl/2 for an initial, single dose of 5 ~M is 40/~sec, giving a Cot value of 1.5 × 10-l° M.sec. Thus, less than 4% of the Cot required for footprinting accrues in the first tin. Because the accumulated dose for each tl/2 is equal, over 25 ta/2 values must elapse to accumulate an equivalent dose. Because the tl/2 value doubles after every hi2, it takes 160 msec to achieve 50% of the Cot value of the synchrotron dose. In fact, even if initial [.OH] of several mM could be generated instantaneously, nucleic acid cleavage equivalent to beamline X-9A could not be achieved in 20 msec. The discussion now returns to the electron bunch structure of the synchrotron and its quasicontinuous wave nature. The synchrotron delivers a constant and reproducible 200 nM [.OH]. Reproducible nucleic acid cleavage is essential to quantitative footprinting. In considering a source for radiolysis, the repetition rate should be kHz or higher to assure a sufficient and reproducible dose. Other disadvantages of the Van de Graft (and many other "single shot" sources) are the high energy of the source (3 MeV for the Brookhaven Van de Graft), requiring the use of longer path lengths (cm versus ram) and thus more biological material (ml versus txl). Also, the "peak to peak" intensity of the device varies considerably, making it difficult to assure an equivalent dose for each time point of a kinetic determination.
Millisecond Exposure Shutter An X-ray shutter capable of millisecond exposures is an important tool for calibrating the exposure of samples to the beamline and for conducting equilibrium experiments. A schematic of the "guillotine shutter" that we designed and use is shown in Fig. 2. The shutter consists of a ¼in. steel plate with an adjustable slit (maximum length 100 ram) that can be dropped along a track by releasing a solenoid from outside the experimental hutch.
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FIG. 2. Schematic representation of the X-ray shutter used for manually exposing samples to the X-ray beam.
When the steel plate is at rest the sample is protected from the X-ray beam. Because the gravitational acceleration is constant, X-ray exposure is readily controlled by the length of the slit. A gas piston can be added to the shutter in order to damp the descent of the plate and extend the range of exposures available. The advantages of this design are simplicity and reliability. Details of the shutter design may be provided on request.
Rapid Mixing X-Ray FootprintingDevice A modified Kin-Tek three-syringe quench-flow device 31 is being used to rapidly mix R N A and Mg 2+ and then exposure the sample mixture to the X-ray beam for the appropriate length of time (Fig. 3). An apparatus of the same type is being used routinely for quench-flow DNase I kinetics studies of protein-DNA interactions, la Although the Kin-Tek apparatus has three drive syringes, only two are absolutely required for X-ray footprinting experiments. However, as will be shown later, the third syringe can be used to mix a radical scavenger with the nucleic acid samples immediately following exposure to the X-ray beam. The other basic requirement for a suitable apparatus is a drive motor whose function and speed can be precisely controlled from a remote location. The modifications required to 31 j. Langowski, C. Urbanke, and E. Schuppe, Anal Biochem. 142, 91 (1984); K. A. Johnson, Methods Enzymol. 134, 677 (1986).
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SteppingMotor I Controller
T
Water Out
-
BufferDrive Syringes
T
B
C
Optional quenchsolution
--tot.
valve
""\
Sample Loops
Buffer Reserve
" "....
/'
~m-RXA
I
I
I
)',,
Buffer Reserve
I
!
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ion Water In chamber
/ ~
Wash inlet
i~.~-
Wash inlet
Exit tube --
Precipitation
3. Schematic representation of the Kin-Tek quench-flow apparatus that has been modified for use in synchrotron X-ray footprinting. FIG.
adapt the Kin-Tek apparatus for X-ray work were the replacement of the sample loop selector with an X-ray exposure chamber and reprogramming of the control software (see later). Placement of a lead shield on the front of the mixer surrounding the exposure chamber minimizes damage to the Plexiglas box surrounding the mixing components. An additional lead shield is used to protect the R N A sample present in its loading syringe from
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scattered radiation (Fig. 3). Siliconizing the flow path of the mixer minimizes absorption of R N A to the walls of its tubes. 32 Several features of this apparatus have demonstrated advantages for the execution of X-ray footprinting experiments that should be looked for in adapting other rapid mixing devices for X-ray beamline use. First, the drive syringes, plumbing, and exposure chamber are all enclosed in a circulating water bath. In addition to providing temperature control, the water bath provides an effective shield against scattered radiation within the experimental hutch. Second, the apparatus can be controlled remotely from outside the experimental hutch by simply making an extension for the cable linking the stepping motor and its controller. Third, modification of the apparatus to accept the X-ray exposure chamber was readily accomplished. Fourth, customization of the control software is straightforward. The major modification to the mixing device required for X-ray footprinting experiments was the insertion of an X-ray transparent "exposure chamber" following the "reaction loop" in the solution flow patch (Fig. 3). A schematic drawing of the exposure chamber is shown in Fig. 4. The 25-/xl chamber is machined out of Lexan with the front window made of X-ray transparent Kapton. The l-ram thickness of the flow cell ensures complete penetration of the sample solution by the X-ray beam. The fittings and tubing required to install the chamber were purchased from the Lee Company (Westbrook, CT). Ten microliters of each of the reactants is loaded into separate 3.5-cm lengths of 0.8-mm internal diameter tubing ("sample loops"). The sample loops intersect at a "T junction" with the 6-cm-long reaction loop that is off sufficient volume to hold the combined volume of the reactants (25/zl). The proximal end of the reaction loop connects with the exposure chamber. A final length of tubing is connected to the distal end of the exposure chamber and exits the apparatus. A second T junction can be placed in the flow train after the exposure chamber should the use of the third syringe be desired to mix the radiolyzed samples with a radical quench solution. The modifications that were made to the mixer required reprogramming of the software controlling the stepping motor in order to conduct the 32 Absorption of nucleic acids to the walls of the tubes has only been observed for sample reaction times greater than 2 rain. The acquisition of time points on the order of minutes is not required for progress curves with millisecond time scales. However, because parts of the Tetrahymena ribozyme fold with time scales on the order of minutes, the acquisition of slow folding data with the rapid mixer is desirable in order to assure the consistency of the fast and slow folding progress curves. Unpublished data have established the consistency of manual and rapid mixing methods for reactions such as the slow folding of the core of the Tetrahymena ribozyme.
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small screws
Front view:
screws from outside the Plexiglas box Exposure chamberdimensions: 1 x 2 x 16 mm (depth x width x length) Top view:
~,,,~~ metal plate
,
'
~ '"'
~ "'"•
'
front of Plexiglasbox Kaptonwindow
o-ring
to the outside
from the mixing"T"
FIG. 4. The X-ray exposure chamber. The chamber is machined out of Lexan. All surfaces contacting the sample are rounded so as to maintain smooth solution flow and to minimize sample loss.
X-ray footprinting protocol that is described later. The modifications to the software provided by KinTek were coded and downloaded from the personal c o m p u t e r being used to control the mixer-using utilities supplied by the manufacturer. The modified control software may be provided on request.
Calibrating Volumes of Rapid Mixing Device The general procedures for calibrating the flow rates and volumes of the Kin T e k mixer are provided in the documentation provided by the m a n u f a c t u r e r and have been published. 11 Briefly, the sample loops volumes are determined using a solution containing a known amount of radiolabeled material. One of the sample loops is filled with the radiolabeled solution, the valve is then turned so that it is open toward the syringe, and about 250/xl is expelled by pushing on the syringe. By quantitating the amount of radioactive material in the expelled volume, the volume of each of the sample loops, the loading loops, and the mixing loop can be determined. In addition, the amount of solution that is expelled from the rapid mixer following one revolution of the m o t o r (=5000 steps) should be determined at the m o t o r speed of the experiment. This is to calibrate the n u m b e r of steps necessary to push the sample into the reaction loop, up to the exposure chamber, and then to fill the exposure chamber. The position of the solution meniscus can be confirmed visually during the calibration procedure.
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Fro. 5. Schematic of setup in the experimental hutch.
Setting up Beamline for X-Ray Footprinting The simplest and most flexible placement of the equipment in the experimental hutch is to position the rapid mixer behind the manual shutter as close to the front port of the beamline as possible (Fig. 5). This arrangement allows both manual and rapid mixing experiments to be interchangeably conducted simply by locking the shutter in the open position for rapid mixing experiments. Of course, both apparatuses need to be aligned with the X-ray beam. Placing the samples close to the front port of the beamline minimizes the X-ray exposure times by minimizing scattering of the beam by a i r y The control wires for both devices are run out of the hutch through standard ports to the experimental control station. The small size of the equipment facilitates setup and breakdown of the instruments, allowing efficient use of limited periods of beam time.
Achieving Appropriate Cleavage of Nucleic Acids by .OH A number of factors affect the amount of cleavage of a nucleic acid sample and must be controlled and calibrated. The appropriate amount of 33Beam scattering can also be minimizedby the insertion of an argon-filledor vacuum "flight tube" in the path of the beamline in situations where the presence of other equipment precludes placing the shutter and mixer close to the front port.
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nucleic acid cleavage is 10-30%, at which level, on average, each molecule that is cleaved by .OH is cleaved only once. In order to achieve this level of cleavage, a dose-response calibration should be completed under the reaction conditions of the experiment. The following principal factors affect X-ray cleavage of nucleic acids. 1. The flux of X-rays impacting on a sample varies among different beamlines and as a function of the operative beamline parameters. Important characteristics include the amount (mrads) of X-ray radiation incident on the sample. This is partially determined by the proximity of the hutch to the storage ring and/or the presence of focusing optics (no focusing is present on beamline X-9A). The beamline path, including beryllium and or aluminum windows, as well as the distance in air that the X-ray beam travels within the beamline hutch, must be considered (for X-9A, a 0.5-mm Be window and a 100-/xm AI window are present, as well as a 1.5-m air path). The flux of the beam also depends on the "ring current" of a synchrotron ring, which decreases from its maximum level following injection of the storage ring (currently 350 mA at NSLS) because of energy loss by the electrons encountering gas molecules in the ring vacuum. The ring current may decrease by as much as 50% between "fills." Calibration experiments must be performed to determine the appropriate dose for various values of the ring current. 2. Solution conditions that affect nucleic acid cleavage include temperature, pH, and the chemical composition of the buffer. Commonly used buffers such as Tris and HEPES are effective radical scavengers. Their use will dramatically increase the X-ray exposure required to footprint. 15 Phosphate, cacodylate, citrate, and pyrophosphate buffers are good alternative buffers. Glycerol, present in many biological reagent storage buffers, is also a potent radical scavenger; as little as 0.1% (v/v) will inhibit nucleic acid cleavage by 50%. Other radical scavengers include urea and ethanol.
Preparation of RNA and Reagents The standard precautions for handling RNA-containing solutions are followed during all of the steps of the X-ray footprinting protocol. RNasefree reagents and deionized (18 M r ) water are used in the preparation of all R N A samples. Buffers are placed on ice immediately following thawing of stock solutions. The Tetrahymena thermophila L-21 ScaI ribozyme is transcribed with T7 R N A polymerase from plasmid pT7L-21 digested with
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ScaI and purified by Sephadex G-50 chromatography. 34 The R N A is 5' labeled with [y-32p]ATP and polynucleotide kinase and then purified by electrophoresis on a 4% denaturing acrylamide gel following published protocols. 3s The 32p-labeled R N A can be stored in TE buffer at either 4 ° or - 2 0 °. Required Solutions TE buffer: 10 mM Tris-HC1, pH 7.5, 1 mM EDTA Assay buffer: 10 mM sodium cacodylate, pH 7.5; 0.1 mM EDTA, pH 8.0 Magnesium buffer: 10 mM sodium cacodylate, pH 7.5; 0.1 mM EDTA, pH 8.0; 20 mM MgCI2 Magnesium buffer for folded samples: 10 mM sodium cacodylate, pH 7.5; 0.1 mM EDTA, pH 8.0; 10 mM MgCI2 Precipitation solution: 1.5 M sodium acetate, pH 5.0; 0.25 mg/ml tRNA (in TE buffer) TBE buffer: 0.089 M Tris-base, 0.089 M boric acid, 0.002 M EDTA (from 0.5 M EDTA stock solution at pH 8.0) 2× gel-loading buffer: 10 M Urea, 0.2× TBE, 0.1% (w/v) bromphenol blue and 0.1% (w/v) Xylene Cyanol.
Protocol for Manual Mixing X-Ray Footprinting Experiment This manual mixing protocol is used for all experiments that require rapid detection. These experiments include calibration of acid cleavage by X-rays from a beamline, equilibrium titration ments, and kinetics experiments with time scales on the order nutes.
do not nucleic experiof mi-
1. The R N A and Mg 2+ solutions (previously equilibrated at the desired temperature) are mixed by adding 2/zl of 50 mM magnesium buffer to 8/xl of the R N A solution (-30,000 cpm). The tube is then placed sideways in a sample holder 36 behind the shutter. The sample is held to the bottom of the microfuge tube by surface tension. In order to ensure that the entire sample is exposed to the X-ray beam, the surface area of the sample must be smaller in diameter than the beam and the depth of the sample should not exceed 1 mm (although 34 A. J. Zaug, C. A. Grosshans, and T. R. Cech, Biochemistry 27, 8924 (1988). 35 j. A. Latham, A. J. Zaug, and T. R. Cech, Methods Enzymol. 181, 558 (1990). 36 The sample holder can be a temperature-regulated block in order to conduct experiments at temperatures other than ambient.
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this is energy dependent). A 10-t~l sample volume is optimal in the current configuration of NSLS beamline X-9A. 2. The safety procedures are initiated that allow entry of the X-ray beam into the vacated and locked beamline hutch. These procedures require abut 30 sec for completion. 3. The safety shutter of the beamline hutch is enabled, allowing entry of the X-ray beam. A second or two later, the steel plate of the shutter is allowed to drop, exposing the sample to the X-ray beam. The safety shutter is closed immediately following the conclusion of the experiment, turning off the X-ray beam. The operator enters the hutch to retrieve the sample. This procedure minimizes exposure of the nucleic acid sample to scattered X-ray radiation. 4. An equal volume of 2× loading buffer or precipitation buffer and three volumes of absolute ethanol are added to the sample. Samples are stored on ice outside the beamline hutch until the entire experiment is completed before further processing.
Protocol for Rapid Mixing X-Ray Footprinting Experiment The experimental protocol described here was adapted from that developed for quench-flow DNase I footprinting11; the rationale and background that are common to the X-ray and DNase I kinetic footprinting techniques will not be repeated here. A schematic of the rapid mixing device is shown in Fig. 3. An overview of a MgZ+-initiated quench-flow kinetics RNA folding experiment is illustrated in Fig. 6 and is described later. This protocol can be readily modified to accommodate other folding initiators and to conduct protein-nucleic acid binding studies. 1. The circulating water bath for the rapid mixture is turned on and the apparatus is allowed to equilibrate at the desired temperature. Fifty microliters of the assay buffer is placed in the water bath to equilibrate to temperature. The flow train of the mixer is washed sequentially with deionized water and methanol and dried with a vacuum. 2. The drive syringes A and C are loaded with the assay buffer to be used in the experiments. The valves are positioned so that the syringe is open toward the outside feed; once the syringe is filled (with no air bubbles) the valves are moved so that the syringe is open toward the tubing beneath it but not toward the outside. (Syringe B can be loaded with a solution such as 10 mM thiourea to be mixed with the sample immediately on exiting the exposure chamber. An alternative procedure is to place an aliquot of a quench
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PUSH
397 PUSH
7 F1G.6. Schematicrepresentation of the sequence of events required to obtain each experimental data point in a synchrotron X-ray footprinting kinetics experiment.
solution in the collection tube. 37) The apparatus is then primed with the assay buffer by moving the motor so that it touches both syringes and the buffer from the syringes has filled all the tubing. 3. One-milliliter disposable syringes (C and D in Fig. 3) are loaded with the R N A sample in assay buffer or magnesium buffer. The magnesium buffer is used to initiate the R N A folding reaction. The assay buffer-containing syringe is used for the control samples in which the unfolded R N A is footprinted. Both syringes remain attached to the outside of the Plexiglas box housing the mixer. The mixer is rinsed and dried as in step 1. 4. The appropriate volumes of the RNA-containing solution and magnesium buffer are pushed into the left and right sample loops, respectively, up to the marks indicating 10/xl (5 mm from the valve). (For the "unfolded control" sample, the R N A is mixed with Mg2+-free 37The -OH cleavage reaction may need to be quenched depending on the time scale of the transition, see earlier. Control experimentsfor the RNA foldingreaction conducted with and without a thiourea quench solution in syringe B were indistinguishable (unpublished data).
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assay buffer.) The syringe containing the RNA is covered with a 5.
6. 7.
8.
9. 10.
lead sleeve to protect it from scattered X-ray radiation. The valves are then turned so that they are open toward the drive syringes. The exit tube is inserted through a microfuge top that has a small hole punched in it. A microfuge tube containing 15/zl precipitation buffer is secured to the end of the exit tube. At this point the investigator exits the beamline hutch and initiates the safety procedures required for enabling the beamline. The timing sequence for the sample is entered into the drive motor controller at this time to inject and mix the samples and subsequently expose them to the X-ray beam. Each injection consists of a "pushpause-push-pause-push-push" timing sequence. The first push mixes the samples. The first pause is the time the reagents are allowed to react. This is the only timing parameter that is changed during an experiment; it is usually incremented to cover the course of the expected duration of the folding reaction. The second push moves the sample into the X-ray exposure chamber. The second pause determines the exposure to the X-ray beam. (The exposure to the X-ray beam is equal to the pause time, plus the time it takes the sample to enter and exit the exposure chamber, about 5 msec at the 180-rpm motor speed utilized). The third push moves the sample out of the exposure chamber. The final push is conducted at a slower velocity in order to minimize splashing of the sample as it is expelled into the microfuge collection tube. The safety shutter of the beamline hutch is enabled, allowing entry of the X-ray beam. The timing sequence for the mixer is initiated and a data point is collected. The safety shutter is closed immediately following the conclusion of the experiment (turning off the X-ray beam), and the operator enters the hutch to retrieve the RNA sample. For samples that react for greater than 10 sec, the timing sequence is initiated prior to enabling the X-ray beam in order to minimize the time that the X-rays are entering the experimental hutch. The controller software has been programmed with a countdown timer that allows the experimenter to know when the sample is poised to enter the exposure chamber. Three volumes (300/xl) of cold ethanol are added to the irradiated sample in order to precipitate the RNA. The mixer is washed and dried as described in step 1. The next collection tube is secured to the exit tube. Step 2 is conducted as necessary. Steps 4-9 are repeated until the desired progress curve has been obtained, as described later.
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Mge+-Initiated RNA-Folding Experiment The following protocol describes in detail the conduct of an experiment to follow the kinetics of the Mg>-dependent folding of the Tetrahymena L-21 ScaI ribozyme. Because beam time may be limited, it is essential that the integrity of the biological reagents be checked prior to conducting a synchrotron X-ray footprinting experiment.
Sample and Reagent Preparation 11. Prepare 20-30 ml of the assay buffer, which will be used to fill the drive syringes (5 ml each) and for MgZ+-free samples that do not require magnesium. 12. Precipitation buffer (15/zl) is added to the desired number of siliconized 1.5-ml microfuge tubes. Twenty-eight samples are prepared for a typical folding experiment. 13. Approximately 1.5 × 106 cpm of the [32p]RNA to be used in the experiment is taken from a stock solution (stored at 4°), heated for 1 min at 90°, and then placed in a 42° water bath for 15 min. Thirty to fifty thousand cpm of the [32p]RNA is used for each reaction. The [32p]RNA is diluted in assay buffer to a total volume of 480 /zl. An equal amount of the magnesium buffer is also prepared at this time. These volumes are in excess of what is required for a 28sample experiment to allow for unforeseen events and for loss of sample during the cleaning procedure. 14. Folded R N A (120/zl) is prepared by adding 1.2/xl of 1 M MgC12 to an aliquot of the sample solution prepared in the preceding step. This control sample is incubated at 42 ° for the duration of the experiment (see step 18). A solution of 10 mM MgC12 (120/~1) is also prepared at this time. 15. Place approximately 30 ml of absolute ethanol on ice. 16. Steps 1-3 are conducted to set up the rapid mixer. 17. The first sample to be footprinted is an unfolded control. Steps 3-10 are conducted for two R N A samples using Mg2+-free assay buffer. Following completion of these samples, the right-hand sample port is washed and dried, a syringe containing magnesium buffer is secured to the inlet port, and the new buffer is loaded into the sample loop. 18. The next samples to be footprinted define the RNA-folding transition. Steps 4-10 are again repetitively conducted using magnesium buffer, incrementing the reaction time for each sample. All other timing parameters remain constant throughout the experiment (see step 7).
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19. Because footprinting defines a transition from an initial to a final state, which is converted to "fractional protection" by numerical methods (described in the next section), it is essential that a significant number of data points be acquired for both the initial and the final portions of the kinetics progress curve. A typical kinetics experiment spans several decades in time scale. 20. The last sample to be footprinted is a folded RNA sample. The RNA and magnesium buffer syringes are removed, and the loops are rinsed and dried as described earlier. The RNA sample prepared in step 14 is mixed with the 10 mM Mg 2+ solution. This sample provides confirmation that the folding reaction has gone to completion. 21. The RNA samples from a complete kinetics footprinting experiment (or a series of experiments) that have been kept on ice outside the beamline hutch are brought back to the laboratory and processed for polyacrylamide gel electrophoresis. 9'1l'15 Dried polyacrylamide gels are exposed to phosphor storage screens and scanned. A Phosphorlmager (Molecular Dynamics, Inc., Sunnyvale, CA) is used in our laboratories for this purpose. Alternative devices are commercially available.
Analysis of X-Ray Footprinting Experiments The intensities of the electrophoretic bands present on the digital autoradiograms are analyzed using the Image Quant (Molecular Dynamics) software provided with our scanner? 8 Although the density changes of individual bands can be quantitated separately, improved precision is obtained by quantitating all of the bands within a "block. ''39 The bands within a block are boxed using the tools available in the software. Another block is defined within each lane as a standard; the standard is a band or bands whose density does not change during the folding reaction. When the blocks of interest have been defined for an experiment, the integrated density of each block is calculated and the results exported to a Microsoft Excel spreadsheet. 4° The integrated density of each folding "block" is divided by 38 A number of commercially available programs can be utilized to analyze the images obtained from the gel electrophoretograms. The most important criterion is the ability to process the full 16-bit density resolution of the scanned images. Detailed descriptions of this analysis procedure have been published. 9 39 m "block" is defined as a group of bands whose densities change equivalentlY as either a function or ligand or time for equilibrium or kinetics experiments, respectivelyY 4o The ImageQuant software is integrated with the Microsoft spreadsheet. Other analysis programs can be substituted through the implementation of the appropriate export and conversion functions.
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that of the standard in the lane in order to normalize for variations in the total density of each lane due to sample loss during precipitation or gel loading. Block density data are paired with the reaction time appropriate for the lane to generate the kinetic progress curves. These operations are facilitated through the application of "macros" within the spreadsheet. This densitometric analysis of the digital image(s) of the electrophoretogram of a kinetic "footprint" experiment yields P i , the "apparent protection." Y, the fractional protection of the binding site, is obtained by fitting data to the coupled equations P = Plower + (Pupper -- Plower) Y
(7a)
Y
(7b)
= 1 -
e TM
where p is the "apparent saturation," P~ow~rand P,pper are the lower and upper limits of the transition curve, ka is the first-order association rate constant, and t is time. The initial analyses of these curves are fit using the Magestic fitting program (Logix Consulting, Inc., www.lgx.com) as a macro within Excel and are scaled to Y using the best-fit values of Pupper and P~..... Further analysis of the progress curves, including global analysis of multiple curves, is conducted using the program NONLIN as has been described. 9'~1 The 65% confidence limits estimated in this manner correspond to approximately one standard deviation. When multiple data sets are analyzed globally, each data set is weighted by the inverse of the square root of the variance of its individual fit. Extensive discussions of the issues that must be considered in conducting and analyzing quantitative nuclease protection experiments have been published. 9
1.2
Io • •
1.0
8
•
•
o---J
•
0.8
•
J
•
]
~'o.~ 0.4 0.2
0.0 0
10
20
30 60
Seconds
FIG. 7. Millisecond RNA folding progress curve of bases 212-215 in the P4 helix in the
Tetrahymena thermophila group I intron. The progress curve is composed of four independent experiments analyzed globally. The open symbols represent the "folded control" in which an RNA sample was preequilibrated with Mg2+ prior to injection into the rapid mixer.
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Representative Result for Mg2+-DependentFolding of Tetrahymena Ribozyme An example of a kinetics progress curve obtained on a millisecond timescale is shown in Fig. 7 for the protection of bases 212-215 of the P4-P6 domain of the Tetrahymenaribozyme. The ability of the synchrotron X-ray footprinting progress curves to resolve subsecond rate constants can be seen readily in this example; experimental data have been obtained for virtually all of the reaction. The progress curve is described by a single exponential within the precision of the experiment. The folding rate of this site is among the fastest measured within this ribozyme (0.9 sec-1), and its protection is due to the interaction of the A bulge in P5a with the P4 helix, one of the first steps in the folding pathway. 41 The protection of the Abulge bases from .OH occurs at the same rate, whereas the protection of other bases, e.g., those that make contact with the P9 domain, occur at a slower rate (0.4 see-l). Summary The rapid mixing synchrotron X-ray footprinting technique described in this article allows nucleic acid folding and ligand binding reactions to be followed on a millisecond time resolution with single nucleotide resolution. In principle, the change in .OH protection of every nucleotide in a nucleic acid hundreds of nucleotides long can be monitored separately, In addition, a wide range of solution conditions are compatible with the radiolytic generation of -OH. These characteristics of synchrotron X-ray footprinting create opportunities for conducting thermodynamic and kinetic studies of nucleic acids that are both comprehensive and detailed. Kinetic footprinting studies of a number of systems have been initiated by the Center for Synchrotron Biosciences using this technique.
41 B. Sclavi, M. Sullivan, M. R. Chance, M. Brenowitz, and S. A. Woodson, Science 279, 1940 (1998).