- Email: [email protected]
[8] Heavy atom derivatives of RNA
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[8] H e a v y A t o m D e r i v a t i v e s o f R N A
By BARBARA L. GOLDEN Introduction Recently, crystallographic studies of R N A have enjoyed a rebirth. This is due partly to technological advances that allow milligram quantities of RNA to be transcribed in vitro or smaller RNAs to be synthesized. A second advance is the development of sparse matrix approaches, 1-4 which allow rapid identification of crystallization conditions for R N A molecules. As with proteins, there is a second major stumbling block in R N A crystallography: the preparation of suitable heavy atom derivatives of the molecule. These derivatives are essential to the calculation of crystallographic phases and generation of an electron density map when solving new structures. Heavy atom derivatives may be obtained in several ways. Compounds can be soaked into preformed crystals or cocrystallized with the molecule. If there are sites within the molecule that tightly bind to the compound, a useful derivative may be obtained. This is the classical method of heavy atom derivatization and it has been used to solve the majority of the new protein structures. It is not necessarily trivial, however, to obtain derivatives of R N A in this manner. RNAs are polyanionic and therefore can frequently interact with cationic metal compounds in a nonspecific manner. The structure of RNAs is often dependent on the presence of divalent metal ions, usually magnesium, and the magnesium in the crystallization conditions can compete with heavy atom binding. R N A also lacks the library of functional groups present in protein molecules (i.e., the sulfhydryl group of cysteine), thus the type of interactions often involved in heavy atoms binding to proteins are lacking in RNA. Additionally, soaking crystals in heavy atom compounds can be detrimental to the diffraction of the crystals, or alter the packing of the R N A within the crystals, rendering the derivative nonisomorphous with native crystals (although this is less of an issue in the age of MAD phasing). This being said, specific binding sites for soaked-in heavy atom compounds have been found in the P4-P6 domain of the Tetrahymena a j. A. Doudna, C. Grosshans, A. Gooding, and C. E. Kundrot, Proc. Natl. Acad. Sci. U.S.A. 90, 7829 (1993). 2 W. G. Scott, J. T. Finch, R. Grenfell, J. Fogg, T. Smith, M. J. Gait, and A. Klug, J. Mol. Biol. 250, 327 (1995). 3 A. C. Anderson, B. E. Earp, and C. A. Frederick, J. Mol. Biol. 259, 696 (1996). 4 B. L. Golden, E. R. Podell, A. R. Gooding, and T. R. Cech, J. Mol. Biol. 2709 711 (1997).
METHODS IN ENZYMOLOGY, VOL. 317
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group I, 5 a 247-nucleotide ribozyme derived from the same intron, 6 the hammerhead ribozyme, 7 the fragment I domain of 5S rRNA, 8 and various tRNAs. 9 The alternative to this "soak and pray" methodology is engineering heavy atoms or heavy atom binding sites into the covalent structure of the R N A to create derivatives. There are advantages to using engineered derivatives. First, the modification introduced is usually minimal. Therefore the macromolecule often behaves like the native, underivatized, species, and crystals of the derivatized macromolecule often diffract as well as the native crystals. Second, since the heavy atom is present in most of the molecules within the crystal, engineered derivatives often result in tightly bound heavy atoms with high occupancy. Third, the site of the modification within the primary sequence is known. This provides a landmark in the initial interpretation of the electron density maps, a distinct advantage, especially when dealing with lower resolution maps. Engineering of heavy atom binding sites into proteins (i.e., by selenomethionine incorporation 1°) or introduction of cysteine residues to bind mercury 11 is one of the reasons for the explosion in the number of protein crystal structures solved in recent years. R N A crystallography does not yet have such a simple methodology for production of heavy atom derivatives. Much more effort is involved in preparing large RNAs with specific modifications in quantities sufficient for a crystallization experiment. This article explores methods that can be used to incorporate heavy atoms or heavy atom binding sites into RNAs for use as heavy atom derivatives. Included are a review of the modifications suitable for creating useful heavy atom derivatives, strategies available for incorporating the modification into large RNAs, and methods for probing crystalline R N A to help identify sites within the R N A crystal that remain accessible for modification. Bromine or Iodine Derivatives of RNAs The simplest way to create a heavy atom derivative of R N A is to incorporate one of the halogenated pyrimidines in place of its unmodified 5 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). 6 B. L. Golden, A. R. Gooding, E. R. Podell, and T. R. Cech, Science 282, 259 (1998). 7 H. W. Pley, K. M. Flaherty, and D. B. McKay, Nature (Lond.) 372, 68 (1994). 8 C. C. Correll, B. Freeborn, P. B. Moore, and T. A. Steitz, Cell 91, 705 (1997). 9 S.-H. Kim, W.-C. Shin, and R. W. Warrant, Methods Enzyrnol. 114, 156 (1985). 10W. A. Hendrickson, J. R. Horton, and D. M. LeMaster, EMBO J. 9, 1665 (1990). 11 G. F. Hatfull, M. R. Sanderson, P. S. Freemont, P. R. Raccuia, N. D. F. Grindley, and T. A. Steitz, Z Mol. Biol. 208, 661 (1989).
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counterpart. Bromine or iodine substitution at the 5 position of uracil or cytosine residues is an often used modification. Halogenation of R N A for use as heavy atom derivatives is a classical method used in the investigation of tRNA structures. 12'13 Bromine derivatives have the advantage of an anomalous diffraction signal that can be harnessed, using a synchrotron radiation source, to provide additional phase information. The higher electron density of an iodine atom, however, may make initial localization of heavy atom positions within the unit cell easier, especially when using a rotating enode X-ray source. This may be the more important consideration when working with large RNAs. The most common means of incorporating bromine or iodine is to chemically synthesize the RNAs and incorporate them in the synthesis. Reagents for making all of these modifications are available commercially. Chemically synthesized, modified RNAs have provided useful halogenated derivatives of the hammerhead ribozyme,7'14 a pseudoknot, 15and numerous RNA duplexes. 16'17 If carefully designed, the modification may also be introduced by transcription. For example, if there are only one or two uridines in this fragment, transcription in the presence of bromouridine triphosphate in place of uridine triphosphate may provide a suitable modification. 18 However, as multiple modifications are introduced, each modification has the potential to interfere with R N A structure or crystal contacts. The all-or-nothing nature of this method may make it less useful in the long run. When working with halogenated RNAs, it is important to protect the sample from light during purification, crystallization, and, if possible, even during data collection to maximize the occupancy of the heavy atom.
Mercury Derivatives of RNA An alternate method of derivatization may be accomplished by incorporating a mercury-binding site into a R N A via a phosphorothioate in the
12 M. Pasek, M. P. Venkatappa, and P. B. Sigler, Biochemistry 12, 4834 (1973). 13j. Tropp and P. B. Sigler, Biochemistry 18, 5489 (1979). 14W. G. Scott, J. T. Finch, and A. Klug, Cell 81, 991 (1995). 15 S. E. Lietzke, C. L. Barnes, V. F. Malone, J. T. Jones, and C. E. Kundrot, in "Structure, Motion, Interaction and Expression of Biological Maeromolecules, Proceedings of the Tenth Conversation" (R. H. Sarma and M. H. Sarma, eds.), p. 91. State University of New York, Albany, 1997. 16 S. E. Lietzke, C. L. Barnes, J. A. Berglund, and C. E. Kundrot, Structure 4, 917 (1996). 17 C. C. Correll, A. Munishkin, Y. L. Chart, Z. Ren, I. G. Wool, and T. A. Steitz, Proc. Natl. Acad. Sci. U.S.A. 95, 13436 (1998). is L. Su, L. Chen, M. Egli, J. M. Berger, and A. Rich, Nature Struct. Biol. 6, 285 (1999).
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R N A backbone. This technique has been used to solve crystal structures of DNA-protein complexes (see, for example, Refs. 19 and 20) and, in theory, it should prove equally powerful in solving R N A structures. However it has not yet been used to successfully derivatize a R N A molecule (see Ref. 21). There are distinct advantages to mercury as a derivative. Mercury is heavy enough to be easily located within the unit cell using Patterson maps, and it has an anomalous scattering signal that can be harnessed to provide additional phasing information. Thus, it may be very helpful in solving the structure of larger RNAs. Technically this method is more complicated than use of halogenated pyrimidines in that mercuration is an additional step in the process, and conditions for mercurating the crystal may need to be explored to optimize the usefulness of the heavy atom derivative. Both Rp and Sp isomers are made during chemical synthesis of phosphorothioate-containing RNAs, and these must often be separated prior to crystallization (see, for examples, Refs. 21 and 22). To minimize interference by the modification and to maximize the occupancy of the heavy atom, independent crystallization and derivatization trials need to be performed on each isomer. Additionally, the nucleotide 5' to the modification should be synthesized with a deoxyribose to prevent loss of the sulfur atom during mercury treatment. Mercury can be bound to the sulfur either prior to crystallization or soaked into preformed crystals, and both should be tried. The mercury atom can be provided as an inorganic compound such as mercury chloride or mercury acetate, or as an organic mercurial compound such as methylmercury chloride or ethylmercury phosphate. The latter compounds have the advantage that they have only one labile ligand and therefore cannot cross-link two modified R N A strands. If the R N A is to be mercurated after crystallization, the mercury compound is introduced into crystallization solutions, typically at concentrations between 0.1 and 5 mM. Another rarely explored option is direct mercuration at the C-5 position of pyrimidines. 21'23 This can be done by reaction for 3-4 hr at 50° with a molar excess of mercury acetate in acetate buffer at pH 5-6. All of the pyrimidines within an oligonucleotide are going to be subject to mercuration by this methodology. Thus, the usefulness of this approach may be limited.
19W. Yang and T. A. Steitz, Cell 82, 193 (1995). 2oM. P. Horvath, V. L. Schweiker, J. M. Bevilacqua, J. A. Ruggles, and S. C. Schultz, Cell 95, 963 (1998). 21 C. C. Correll, B. Freeborn, P. B. Moore, and T. A. Steitz, J. Biomol. Struct. Dynamics 15, 165 (1997). 22 E. C. Scott and O. C. Uhlenbeck, Nucleic Acids Res. 27, 479 (1999). 23 R. M. K. Dale, D. C. Livingston, and D. C. Ward, Proc. Natl. Acad. Sci. U.S.A. 70, 2238 (1973).
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Site Selection The first consideration when engineering a heavy atom binding site into the R N A is to refrain from disturbing interactions required for proper folding of the R N A and for formation of crystal contacts. Halogenated pyrimidines alter the functional groups in the major groove and modification of the phosphate oxygen atoms is largely going to affect the major groove as well. Thus, both modifications steer clear of the shallow minor groove crucial for many tertiary interactions within R N A structures. Within the P 4 - P 6 domain, the best derivatives were obtained by modification of uridines involved in G . U wobble pairs. 24 It will be interesting to see if this continues to be a trend. While some thought should go into deciding where the effects of the modification will be minimized, this is always going to be somewhat of a trial-and-error proposition. Where biochemical assays are available to report on the structure, it may be worthwhile to make the modified R N A in biochemical quantities first, and scale up those modifications that pass this preliminary test.
Engineering Large RNAs If the R N A under investigation is too long to be made in quantity by chemical synthesis, than engineering a heavy atom derivative becomes more difficult. Modification of RNAs greater than - 3 0 nucleotides requires that the R N A be made in two pieces, one carries the modification, and the second, which can be made by in vitro transcription, makes up the remainder of the molecule. These two pieces must then be reconstituted somehow prior to crystallization to make the full-length product. One means of reconstitution is to covalently join the two pieces using T4 D N A ligase and a D N A splintY This method requires that the R N A corresponding to the 5' end has a free 3'-hydroxyl group, and the R N A corresponding to the 3' end possess a 5' phosphate. Phosphorylation at the 5' end can be accomplished by transcription in the presence of 10-fold molar excess of G M P over G T P or by T4 polynucleotide kinase. Synthetic RNAs can be phosphorylated enzymatically using T4 polynucleotide kinase or chemically during the synthesis. The two RNAs are then mixed with a D N A oligonucleotide which is complementary to the junction to be joined, and usually heated and cooled to anneal the three oligonucleotides. This forms a D N A - R N A heteroduplex with a nick at the junction of the two RNAs. If this structure is correctly formed, T4 ligase is capable of covalently linking the two RNAs. 24B. L. Golden, A. R. Gooding, E. R. Podell, and T. R. Cech, RNA 2, 1295 (1996). 25M. J. Moore and P. A. Sharp, Science 256, 992 (1992).
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A typical ligation reaction might contain the following: 60-70 mM Tris-HC1, pH 7.8-8.2 10 mM MgC12 10 mM Dithiothreitol (DT-F) 0.5-1.0 mM adenosine triphosphate (ATP) 20/zM each RNA 20/xM DNA splint 50-250 units//.d T4 DNA ligase This reaction can be performed at room temperature or at 16°, which is the temperature at which the ligase possesses maximal activity. To maximize the efficiency of the reaction with a minimum of ligase, the reaction can be run over the course of several hours or even overnight. It is important to design the system such that the ligation reaction is efficient. Poor yields in the ligation reaction can result if a strong secondary or tertiary structure interferes with proper annealing of the splint. Ligation efficiency should be checked by performing small-scale reactions with unmodified RNAs prior to undertaking large-scale reactions. It may be necessary to explore several variations to find a system that allows efficient ligation. Several steps may be taken to increase a low yield. Most of these involve changing the annealing protocol. Band shift analysis may be used to monitor annealing of the three oligonucleotides independent of the ligation reaction. Annealing can be accomplished by heating the oligos to 95° and snap cooling on ice. Alternately the molecules may be slowly cooled from 95° to room temperature in a heating block or a using a thermal cycle. Other systems have benefited from heating for longer time periods at lower temperatures. The order of addition of the three components can be crucial; it may be necessary to anneal the DNA to one RNA molecule, then reanneal in the presence of the second RNA. Occasionally redesigning the DNA splint has had dramatic effects. Addition of one or two extra complementary nucleotides to the end of the splint can help in formation of the ternary complex, perhaps due to disruption of residual secondary structure in the RNA. Also along these lines, a second DNA oligonucleotide complementary to the RNA at a distal site may be added to the annealing reaction to disrupt secondary structure within the RNA during ligation reaction. 26 Sodium chloride (up to 20 mM) may be added to the buffer to increase the efficiency of the annealing reaction, however higher concentrations ( - 5 0 mM) will inhibit T4 ligase. As an alternative to ligation, the two pieces can be simply be annealed by base pairing and tertiary interactions without covalent ligation, creating a nicked version of the crystallized molecule.24 This method has the benefit that optimization of the ligation reaction is not necessary, however, the 26 S. A. Strobel and T. R. Cech, Comments 19, 89 (1992).
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nick introduced cannot profoundly destabilize the overall structure or else crystallization will be effected. The two options are somewhat complementary. If the RNA has strong secondary and tertiary structure, residual structure in the RNA can interfere with the ligation reaction. The same strong tertiary structure often results in stable folding of a nicked species of the RNA. This may allow assembly of the two pieces without covalent ligation. Assembly can be assayed by native gel-shift analysis or by biochemical assays. The trick to using either of these approaches is to find a site within 30 nucleotides of either the 5' or 3' terminus that will allow efficient ligation or, in the case of the latter method, will tolerate the introduction of a break in the backbone. The use of cis- or trans-acting ribozymes (often hammerhead sequences) may be very useful in the design of these two-piece systems.24Posttranscriptional processing of RNAs has several advantages. First, if a deletion is to be made at the 5' end of a transcript, the new 5' end must retain a sequence that allows efficient T7 transcription, usually this sequence begins with a guanosine and is purine rich. 27 This is an additional constraint that may be difficult to work around. If a hammerhead sequence is incorporated at the 5' end of the transcript, however, the efficient T7 start site precedes the hammerhead and thus is cleaved off during posttranscriptional processing. The sequence at the 5' end of the final product has no sequence constraints, and possesses a free 5'-hydroxyl group that is readily phosphorylated using T4 polynucleotide kinase. This strategy allows more flexibility in situations where the smaller oligonucleotide is to be added at the 5' end of the molecule. If the smaller oligonucleotide is to be added to the 3' end of the molecule, hammerhead processing of the 3' end may or may not be advisable. On one hand, this technique eliminates heterogeneity at the 3' end characteristic of T7 polymerase transcripts. This is important if this RNA is to be ligated because the uncoded nucleotides added to a portion of the T7 transcripts will interfere with the ligation reaction. However, hammerhead processing imposes some restriction on the sequence--specifically the 3'-terminal residue may not be a guanosine. Additionally, the cleavage product of the hammerhead results in a 3' end that is blocked with a 2',3'-cyclic phosphate which must be removed if the product is to be covalently joined to another RNA by T4 ligase. Analysis of Crystallized RNA Biochemical characterization of crystalline RNA can provide strategies for the introduction of modifications. During the crystallization process, 27j. F. Milliganand O. C. Uhlenbeck,Methods Enzymol. 180,51 (1989).
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the R N A within the crystals often accumulates nicks. R N A obtained from redissolved crystals can be reverse transcribed or labeled at the 5' end with [~/-32p]ATP using T4 polynucleotide kinase. Breaks in the R N A are revealed as strong stops not present in freshly prepared RNA. If nicks are present within 30 nucleotides of either terminus, this provides strong evidence that it will be possible to cocrystallize two appropriately designed RNAs without prior ligation. It may also be possible to identify and avoid sites that are involved in crystal contacts by chemical modification of R N A within the crystal. Dimethylsulfate (DMS) mapping of crystallized R N A was used to successfully predict a crystal contact in the P5c loop of the P4-P6 domain. 28 Comparison of the modification pattern in the crystal and in solution may also reveal formation of nonnative structures resulting from crystal packing prior to actually solving the structure. Crystallization of nonnative structures is a continuing problem in R N A crystallography. 15,29,3° To perform analyses on crystallized RNA, a suitable stabilizing buffer must first be identified that will not dissolve or crack the crystals. This stabilizing buffer usually contains precipitant, buffer, magnesium, polyamines, and other salts, but no RNA, and it will be different for every crystal form. By carefully washing crystals with the buffer prior to dissolving them, crystallized R N A can be selectively isolated from the drop. Thus, the analysis will correspond only to the R N A incorporated in the crystal and not the molecules that remain soluble during the crystallization process. The Tetrahymena ribozyme c r y s t a l s , 4 which are ~70% solvent and - 2 0 0 400/zm in each dimension, are calculated to have - 4 mM R N A within the crystal (1 M nucleotides), and to contain ~1.0/zg of R N A each. This is going to vary somewhat depending on the solvent content of the crystal and the size of the RNA, but in general, one medium-sized crystal will be more than sufficient to allow optimization of reverse transcription experiments. Crystals that are poorly formed and therefore unsuitable for a diffraction experiment are fine for these analyses. To examine the DMS reaction pattern of the R N A within the crystal, a procedure similar to that used by Zaug and Cech 31 is used. Prior to modification, crystals are washed several times in stabilization buffer. The crystals are then modified by treatment with stabilization buffer that contains 10% DMS stock solution (2 ~1 DMS, 7/zl absolute ethanol). Using a different crystal for each experiment, several time points (15 min to several 2s B. L. Golden and T. R. Cech, unpublished results. 29 S. R. Holbrook, C. Cheong, I. Tinoeo, Jr., and S. H. Kim, Nature (Lond.) 353, 579 (1991). 30 j. Nowakowski, P. J. Shim, G. S. Prasad, C. D. Stout, and G. F. Joyce, Nature Struct. Biol. 6, 151 (1999). 31 A. J. Zaug and T. R. Cech, R N A 1, 363 (1995).
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hours) are observed. The reaction is then quenched by dissolving the crystals in 0.5 volumes of stop solution (0.5 M 2-mercaptoethanol, 0.75 M sodium acetate, pH 5.5). The redissolved RNA can then be ethanol precipitated by addition of 3 volumes of ethanol, and resuspended in 100/.d volume. Under ideal conditions, modification of the RNA is readily apparent in the analysis that followed, but the crystal remains visually intact with no cracking or surface striations. Modification patterns are revealed by reverse transcription of the RNA using avian myeloblastosis virus (AMV) reverse transcriptase. 31 Adenosine and cytosine residues accessible to modification are identified as stops in a reverse transcription reaction. These nucleotides can then be identified by comparison with a sequencing ladder. The amount of RNA in a crystal with dimensions of ~200 tzm is sufficient for -100 reverse transcriptase reactions. It is important to compare the modified RNA to unmodified crystalline RNA because nicks in the backbone accumulate during crystallization and these too will appear as stops in the reverse transcription reaction. Fe. EDTA protection analysis might seem to be ideal for this type of analysis, since this reagent reveals regions of the RNA that are inaccessible to the solvent. Presumably regions involved in crystal contacts will be protected from the solvent. However MPD and polyethylene glycol which are often used as precipitants are capable of quenching this free radical formation. It will be interesting to see if crystals grown under high salt conditions can be probed using free-radical reagents. Acknowledgments I am grateful to Tom Cech, Anne Gooding, and Elaine PodeU for help with many of the technical details outlined here. This is paper 15955 from the Purdue Agriculture Experiment station.
[9] Site-Specific Cleavage of Transcript RNA By J O N
L A P H A M a n d D O N A L D M . CROTHERS
Introduction We describe a method by which transcript RNA is cleaved site specifically using ribonuclease H (RNase H) and a targeting 2'-O-methyl-RNA/ DNA chimera. The reaction is high yielding, has no sequence specificity requirements, and has been adapted to the cleavage of large quantities
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[8] H e a v y A t o m D e r i v a t i v e s o f R N A
By BARBARA L. GOLDEN Introduction Recently, crystallographic studies of R N A have enjoyed a rebirth. This is due partly to technological advances that allow milligram quantities of RNA to be transcribed in vitro or smaller RNAs to be synthesized. A second advance is the development of sparse matrix approaches, 1-4 which allow rapid identification of crystallization conditions for R N A molecules. As with proteins, there is a second major stumbling block in R N A crystallography: the preparation of suitable heavy atom derivatives of the molecule. These derivatives are essential to the calculation of crystallographic phases and generation of an electron density map when solving new structures. Heavy atom derivatives may be obtained in several ways. Compounds can be soaked into preformed crystals or cocrystallized with the molecule. If there are sites within the molecule that tightly bind to the compound, a useful derivative may be obtained. This is the classical method of heavy atom derivatization and it has been used to solve the majority of the new protein structures. It is not necessarily trivial, however, to obtain derivatives of R N A in this manner. RNAs are polyanionic and therefore can frequently interact with cationic metal compounds in a nonspecific manner. The structure of RNAs is often dependent on the presence of divalent metal ions, usually magnesium, and the magnesium in the crystallization conditions can compete with heavy atom binding. R N A also lacks the library of functional groups present in protein molecules (i.e., the sulfhydryl group of cysteine), thus the type of interactions often involved in heavy atoms binding to proteins are lacking in RNA. Additionally, soaking crystals in heavy atom compounds can be detrimental to the diffraction of the crystals, or alter the packing of the R N A within the crystals, rendering the derivative nonisomorphous with native crystals (although this is less of an issue in the age of MAD phasing). This being said, specific binding sites for soaked-in heavy atom compounds have been found in the P4-P6 domain of the Tetrahymena a j. A. Doudna, C. Grosshans, A. Gooding, and C. E. Kundrot, Proc. Natl. Acad. Sci. U.S.A. 90, 7829 (1993). 2 W. G. Scott, J. T. Finch, R. Grenfell, J. Fogg, T. Smith, M. J. Gait, and A. Klug, J. Mol. Biol. 250, 327 (1995). 3 A. C. Anderson, B. E. Earp, and C. A. Frederick, J. Mol. Biol. 259, 696 (1996). 4 B. L. Golden, E. R. Podell, A. R. Gooding, and T. R. Cech, J. Mol. Biol. 2709 711 (1997).
METHODS IN ENZYMOLOGY, VOL. 317
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group I, 5 a 247-nucleotide ribozyme derived from the same intron, 6 the hammerhead ribozyme, 7 the fragment I domain of 5S rRNA, 8 and various tRNAs. 9 The alternative to this "soak and pray" methodology is engineering heavy atoms or heavy atom binding sites into the covalent structure of the R N A to create derivatives. There are advantages to using engineered derivatives. First, the modification introduced is usually minimal. Therefore the macromolecule often behaves like the native, underivatized, species, and crystals of the derivatized macromolecule often diffract as well as the native crystals. Second, since the heavy atom is present in most of the molecules within the crystal, engineered derivatives often result in tightly bound heavy atoms with high occupancy. Third, the site of the modification within the primary sequence is known. This provides a landmark in the initial interpretation of the electron density maps, a distinct advantage, especially when dealing with lower resolution maps. Engineering of heavy atom binding sites into proteins (i.e., by selenomethionine incorporation 1°) or introduction of cysteine residues to bind mercury 11 is one of the reasons for the explosion in the number of protein crystal structures solved in recent years. R N A crystallography does not yet have such a simple methodology for production of heavy atom derivatives. Much more effort is involved in preparing large RNAs with specific modifications in quantities sufficient for a crystallization experiment. This article explores methods that can be used to incorporate heavy atoms or heavy atom binding sites into RNAs for use as heavy atom derivatives. Included are a review of the modifications suitable for creating useful heavy atom derivatives, strategies available for incorporating the modification into large RNAs, and methods for probing crystalline R N A to help identify sites within the R N A crystal that remain accessible for modification. Bromine or Iodine Derivatives of RNAs The simplest way to create a heavy atom derivative of R N A is to incorporate one of the halogenated pyrimidines in place of its unmodified 5 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). 6 B. L. Golden, A. R. Gooding, E. R. Podell, and T. R. Cech, Science 282, 259 (1998). 7 H. W. Pley, K. M. Flaherty, and D. B. McKay, Nature (Lond.) 372, 68 (1994). 8 C. C. Correll, B. Freeborn, P. B. Moore, and T. A. Steitz, Cell 91, 705 (1997). 9 S.-H. Kim, W.-C. Shin, and R. W. Warrant, Methods Enzyrnol. 114, 156 (1985). 10W. A. Hendrickson, J. R. Horton, and D. M. LeMaster, EMBO J. 9, 1665 (1990). 11 G. F. Hatfull, M. R. Sanderson, P. S. Freemont, P. R. Raccuia, N. D. F. Grindley, and T. A. Steitz, Z Mol. Biol. 208, 661 (1989).
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counterpart. Bromine or iodine substitution at the 5 position of uracil or cytosine residues is an often used modification. Halogenation of R N A for use as heavy atom derivatives is a classical method used in the investigation of tRNA structures. 12'13 Bromine derivatives have the advantage of an anomalous diffraction signal that can be harnessed, using a synchrotron radiation source, to provide additional phase information. The higher electron density of an iodine atom, however, may make initial localization of heavy atom positions within the unit cell easier, especially when using a rotating enode X-ray source. This may be the more important consideration when working with large RNAs. The most common means of incorporating bromine or iodine is to chemically synthesize the RNAs and incorporate them in the synthesis. Reagents for making all of these modifications are available commercially. Chemically synthesized, modified RNAs have provided useful halogenated derivatives of the hammerhead ribozyme,7'14 a pseudoknot, 15and numerous RNA duplexes. 16'17 If carefully designed, the modification may also be introduced by transcription. For example, if there are only one or two uridines in this fragment, transcription in the presence of bromouridine triphosphate in place of uridine triphosphate may provide a suitable modification. 18 However, as multiple modifications are introduced, each modification has the potential to interfere with R N A structure or crystal contacts. The all-or-nothing nature of this method may make it less useful in the long run. When working with halogenated RNAs, it is important to protect the sample from light during purification, crystallization, and, if possible, even during data collection to maximize the occupancy of the heavy atom.
Mercury Derivatives of RNA An alternate method of derivatization may be accomplished by incorporating a mercury-binding site into a R N A via a phosphorothioate in the
12 M. Pasek, M. P. Venkatappa, and P. B. Sigler, Biochemistry 12, 4834 (1973). 13j. Tropp and P. B. Sigler, Biochemistry 18, 5489 (1979). 14W. G. Scott, J. T. Finch, and A. Klug, Cell 81, 991 (1995). 15 S. E. Lietzke, C. L. Barnes, V. F. Malone, J. T. Jones, and C. E. Kundrot, in "Structure, Motion, Interaction and Expression of Biological Maeromolecules, Proceedings of the Tenth Conversation" (R. H. Sarma and M. H. Sarma, eds.), p. 91. State University of New York, Albany, 1997. 16 S. E. Lietzke, C. L. Barnes, J. A. Berglund, and C. E. Kundrot, Structure 4, 917 (1996). 17 C. C. Correll, A. Munishkin, Y. L. Chart, Z. Ren, I. G. Wool, and T. A. Steitz, Proc. Natl. Acad. Sci. U.S.A. 95, 13436 (1998). is L. Su, L. Chen, M. Egli, J. M. Berger, and A. Rich, Nature Struct. Biol. 6, 285 (1999).
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R N A backbone. This technique has been used to solve crystal structures of DNA-protein complexes (see, for example, Refs. 19 and 20) and, in theory, it should prove equally powerful in solving R N A structures. However it has not yet been used to successfully derivatize a R N A molecule (see Ref. 21). There are distinct advantages to mercury as a derivative. Mercury is heavy enough to be easily located within the unit cell using Patterson maps, and it has an anomalous scattering signal that can be harnessed to provide additional phasing information. Thus, it may be very helpful in solving the structure of larger RNAs. Technically this method is more complicated than use of halogenated pyrimidines in that mercuration is an additional step in the process, and conditions for mercurating the crystal may need to be explored to optimize the usefulness of the heavy atom derivative. Both Rp and Sp isomers are made during chemical synthesis of phosphorothioate-containing RNAs, and these must often be separated prior to crystallization (see, for examples, Refs. 21 and 22). To minimize interference by the modification and to maximize the occupancy of the heavy atom, independent crystallization and derivatization trials need to be performed on each isomer. Additionally, the nucleotide 5' to the modification should be synthesized with a deoxyribose to prevent loss of the sulfur atom during mercury treatment. Mercury can be bound to the sulfur either prior to crystallization or soaked into preformed crystals, and both should be tried. The mercury atom can be provided as an inorganic compound such as mercury chloride or mercury acetate, or as an organic mercurial compound such as methylmercury chloride or ethylmercury phosphate. The latter compounds have the advantage that they have only one labile ligand and therefore cannot cross-link two modified R N A strands. If the R N A is to be mercurated after crystallization, the mercury compound is introduced into crystallization solutions, typically at concentrations between 0.1 and 5 mM. Another rarely explored option is direct mercuration at the C-5 position of pyrimidines. 21'23 This can be done by reaction for 3-4 hr at 50° with a molar excess of mercury acetate in acetate buffer at pH 5-6. All of the pyrimidines within an oligonucleotide are going to be subject to mercuration by this methodology. Thus, the usefulness of this approach may be limited.
19W. Yang and T. A. Steitz, Cell 82, 193 (1995). 2oM. P. Horvath, V. L. Schweiker, J. M. Bevilacqua, J. A. Ruggles, and S. C. Schultz, Cell 95, 963 (1998). 21 C. C. Correll, B. Freeborn, P. B. Moore, and T. A. Steitz, J. Biomol. Struct. Dynamics 15, 165 (1997). 22 E. C. Scott and O. C. Uhlenbeck, Nucleic Acids Res. 27, 479 (1999). 23 R. M. K. Dale, D. C. Livingston, and D. C. Ward, Proc. Natl. Acad. Sci. U.S.A. 70, 2238 (1973).
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Site Selection The first consideration when engineering a heavy atom binding site into the R N A is to refrain from disturbing interactions required for proper folding of the R N A and for formation of crystal contacts. Halogenated pyrimidines alter the functional groups in the major groove and modification of the phosphate oxygen atoms is largely going to affect the major groove as well. Thus, both modifications steer clear of the shallow minor groove crucial for many tertiary interactions within R N A structures. Within the P 4 - P 6 domain, the best derivatives were obtained by modification of uridines involved in G . U wobble pairs. 24 It will be interesting to see if this continues to be a trend. While some thought should go into deciding where the effects of the modification will be minimized, this is always going to be somewhat of a trial-and-error proposition. Where biochemical assays are available to report on the structure, it may be worthwhile to make the modified R N A in biochemical quantities first, and scale up those modifications that pass this preliminary test.
Engineering Large RNAs If the R N A under investigation is too long to be made in quantity by chemical synthesis, than engineering a heavy atom derivative becomes more difficult. Modification of RNAs greater than - 3 0 nucleotides requires that the R N A be made in two pieces, one carries the modification, and the second, which can be made by in vitro transcription, makes up the remainder of the molecule. These two pieces must then be reconstituted somehow prior to crystallization to make the full-length product. One means of reconstitution is to covalently join the two pieces using T4 D N A ligase and a D N A splintY This method requires that the R N A corresponding to the 5' end has a free 3'-hydroxyl group, and the R N A corresponding to the 3' end possess a 5' phosphate. Phosphorylation at the 5' end can be accomplished by transcription in the presence of 10-fold molar excess of G M P over G T P or by T4 polynucleotide kinase. Synthetic RNAs can be phosphorylated enzymatically using T4 polynucleotide kinase or chemically during the synthesis. The two RNAs are then mixed with a D N A oligonucleotide which is complementary to the junction to be joined, and usually heated and cooled to anneal the three oligonucleotides. This forms a D N A - R N A heteroduplex with a nick at the junction of the two RNAs. If this structure is correctly formed, T4 ligase is capable of covalently linking the two RNAs. 24B. L. Golden, A. R. Gooding, E. R. Podell, and T. R. Cech, RNA 2, 1295 (1996). 25M. J. Moore and P. A. Sharp, Science 256, 992 (1992).
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A typical ligation reaction might contain the following: 60-70 mM Tris-HC1, pH 7.8-8.2 10 mM MgC12 10 mM Dithiothreitol (DT-F) 0.5-1.0 mM adenosine triphosphate (ATP) 20/zM each RNA 20/xM DNA splint 50-250 units//.d T4 DNA ligase This reaction can be performed at room temperature or at 16°, which is the temperature at which the ligase possesses maximal activity. To maximize the efficiency of the reaction with a minimum of ligase, the reaction can be run over the course of several hours or even overnight. It is important to design the system such that the ligation reaction is efficient. Poor yields in the ligation reaction can result if a strong secondary or tertiary structure interferes with proper annealing of the splint. Ligation efficiency should be checked by performing small-scale reactions with unmodified RNAs prior to undertaking large-scale reactions. It may be necessary to explore several variations to find a system that allows efficient ligation. Several steps may be taken to increase a low yield. Most of these involve changing the annealing protocol. Band shift analysis may be used to monitor annealing of the three oligonucleotides independent of the ligation reaction. Annealing can be accomplished by heating the oligos to 95° and snap cooling on ice. Alternately the molecules may be slowly cooled from 95° to room temperature in a heating block or a using a thermal cycle. Other systems have benefited from heating for longer time periods at lower temperatures. The order of addition of the three components can be crucial; it may be necessary to anneal the DNA to one RNA molecule, then reanneal in the presence of the second RNA. Occasionally redesigning the DNA splint has had dramatic effects. Addition of one or two extra complementary nucleotides to the end of the splint can help in formation of the ternary complex, perhaps due to disruption of residual secondary structure in the RNA. Also along these lines, a second DNA oligonucleotide complementary to the RNA at a distal site may be added to the annealing reaction to disrupt secondary structure within the RNA during ligation reaction. 26 Sodium chloride (up to 20 mM) may be added to the buffer to increase the efficiency of the annealing reaction, however higher concentrations ( - 5 0 mM) will inhibit T4 ligase. As an alternative to ligation, the two pieces can be simply be annealed by base pairing and tertiary interactions without covalent ligation, creating a nicked version of the crystallized molecule.24 This method has the benefit that optimization of the ligation reaction is not necessary, however, the 26 S. A. Strobel and T. R. Cech, Comments 19, 89 (1992).
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nick introduced cannot profoundly destabilize the overall structure or else crystallization will be effected. The two options are somewhat complementary. If the RNA has strong secondary and tertiary structure, residual structure in the RNA can interfere with the ligation reaction. The same strong tertiary structure often results in stable folding of a nicked species of the RNA. This may allow assembly of the two pieces without covalent ligation. Assembly can be assayed by native gel-shift analysis or by biochemical assays. The trick to using either of these approaches is to find a site within 30 nucleotides of either the 5' or 3' terminus that will allow efficient ligation or, in the case of the latter method, will tolerate the introduction of a break in the backbone. The use of cis- or trans-acting ribozymes (often hammerhead sequences) may be very useful in the design of these two-piece systems.24Posttranscriptional processing of RNAs has several advantages. First, if a deletion is to be made at the 5' end of a transcript, the new 5' end must retain a sequence that allows efficient T7 transcription, usually this sequence begins with a guanosine and is purine rich. 27 This is an additional constraint that may be difficult to work around. If a hammerhead sequence is incorporated at the 5' end of the transcript, however, the efficient T7 start site precedes the hammerhead and thus is cleaved off during posttranscriptional processing. The sequence at the 5' end of the final product has no sequence constraints, and possesses a free 5'-hydroxyl group that is readily phosphorylated using T4 polynucleotide kinase. This strategy allows more flexibility in situations where the smaller oligonucleotide is to be added at the 5' end of the molecule. If the smaller oligonucleotide is to be added to the 3' end of the molecule, hammerhead processing of the 3' end may or may not be advisable. On one hand, this technique eliminates heterogeneity at the 3' end characteristic of T7 polymerase transcripts. This is important if this RNA is to be ligated because the uncoded nucleotides added to a portion of the T7 transcripts will interfere with the ligation reaction. However, hammerhead processing imposes some restriction on the sequence--specifically the 3'-terminal residue may not be a guanosine. Additionally, the cleavage product of the hammerhead results in a 3' end that is blocked with a 2',3'-cyclic phosphate which must be removed if the product is to be covalently joined to another RNA by T4 ligase. Analysis of Crystallized RNA Biochemical characterization of crystalline RNA can provide strategies for the introduction of modifications. During the crystallization process, 27j. F. Milliganand O. C. Uhlenbeck,Methods Enzymol. 180,51 (1989).
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the R N A within the crystals often accumulates nicks. R N A obtained from redissolved crystals can be reverse transcribed or labeled at the 5' end with [~/-32p]ATP using T4 polynucleotide kinase. Breaks in the R N A are revealed as strong stops not present in freshly prepared RNA. If nicks are present within 30 nucleotides of either terminus, this provides strong evidence that it will be possible to cocrystallize two appropriately designed RNAs without prior ligation. It may also be possible to identify and avoid sites that are involved in crystal contacts by chemical modification of R N A within the crystal. Dimethylsulfate (DMS) mapping of crystallized R N A was used to successfully predict a crystal contact in the P5c loop of the P4-P6 domain. 28 Comparison of the modification pattern in the crystal and in solution may also reveal formation of nonnative structures resulting from crystal packing prior to actually solving the structure. Crystallization of nonnative structures is a continuing problem in R N A crystallography. 15,29,3° To perform analyses on crystallized RNA, a suitable stabilizing buffer must first be identified that will not dissolve or crack the crystals. This stabilizing buffer usually contains precipitant, buffer, magnesium, polyamines, and other salts, but no RNA, and it will be different for every crystal form. By carefully washing crystals with the buffer prior to dissolving them, crystallized R N A can be selectively isolated from the drop. Thus, the analysis will correspond only to the R N A incorporated in the crystal and not the molecules that remain soluble during the crystallization process. The Tetrahymena ribozyme c r y s t a l s , 4 which are ~70% solvent and - 2 0 0 400/zm in each dimension, are calculated to have - 4 mM R N A within the crystal (1 M nucleotides), and to contain ~1.0/zg of R N A each. This is going to vary somewhat depending on the solvent content of the crystal and the size of the RNA, but in general, one medium-sized crystal will be more than sufficient to allow optimization of reverse transcription experiments. Crystals that are poorly formed and therefore unsuitable for a diffraction experiment are fine for these analyses. To examine the DMS reaction pattern of the R N A within the crystal, a procedure similar to that used by Zaug and Cech 31 is used. Prior to modification, crystals are washed several times in stabilization buffer. The crystals are then modified by treatment with stabilization buffer that contains 10% DMS stock solution (2 ~1 DMS, 7/zl absolute ethanol). Using a different crystal for each experiment, several time points (15 min to several 2s B. L. Golden and T. R. Cech, unpublished results. 29 S. R. Holbrook, C. Cheong, I. Tinoeo, Jr., and S. H. Kim, Nature (Lond.) 353, 579 (1991). 30 j. Nowakowski, P. J. Shim, G. S. Prasad, C. D. Stout, and G. F. Joyce, Nature Struct. Biol. 6, 151 (1999). 31 A. J. Zaug and T. R. Cech, R N A 1, 363 (1995).
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hours) are observed. The reaction is then quenched by dissolving the crystals in 0.5 volumes of stop solution (0.5 M 2-mercaptoethanol, 0.75 M sodium acetate, pH 5.5). The redissolved RNA can then be ethanol precipitated by addition of 3 volumes of ethanol, and resuspended in 100/.d volume. Under ideal conditions, modification of the RNA is readily apparent in the analysis that followed, but the crystal remains visually intact with no cracking or surface striations. Modification patterns are revealed by reverse transcription of the RNA using avian myeloblastosis virus (AMV) reverse transcriptase. 31 Adenosine and cytosine residues accessible to modification are identified as stops in a reverse transcription reaction. These nucleotides can then be identified by comparison with a sequencing ladder. The amount of RNA in a crystal with dimensions of ~200 tzm is sufficient for -100 reverse transcriptase reactions. It is important to compare the modified RNA to unmodified crystalline RNA because nicks in the backbone accumulate during crystallization and these too will appear as stops in the reverse transcription reaction. Fe. EDTA protection analysis might seem to be ideal for this type of analysis, since this reagent reveals regions of the RNA that are inaccessible to the solvent. Presumably regions involved in crystal contacts will be protected from the solvent. However MPD and polyethylene glycol which are often used as precipitants are capable of quenching this free radical formation. It will be interesting to see if crystals grown under high salt conditions can be probed using free-radical reagents. Acknowledgments I am grateful to Tom Cech, Anne Gooding, and Elaine PodeU for help with many of the technical details outlined here. This is paper 15955 from the Purdue Agriculture Experiment station.
[9] Site-Specific Cleavage of Transcript RNA By J O N
L A P H A M a n d D O N A L D M . CROTHERS
Introduction We describe a method by which transcript RNA is cleaved site specifically using ribonuclease H (RNase H) and a targeting 2'-O-methyl-RNA/ DNA chimera. The reaction is high yielding, has no sequence specificity requirements, and has been adapted to the cleavage of large quantities
METHODS IN ENZYMOLOGY,VOL. 317
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