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A General Module for RNA Crystallization Adrianr.Ferreâ-D'amareâ,Kaihongzhouandjennifera.Doudna*

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A General Module for RNA Crystallization Adrianr.Ferreâ-D'amareâ,Kaihongzhouandjennifera.Doudna* Article No. mb981789 J. Mol. Biol. (1998) 279, 621±631 A General Module for RNA Crystallization AdrianR.FerreÂ-D'AmareÂ,KaihongZhouandJenniferA.Doudna* Department of Molecular Crystallization of RNA molecules other than simple oligonucleotide Biophysics and Biochemistry duplexes remains a challenging step in structure determination by X-ray and Howard Hughes Medical crystallography. Subjecting biochemically, covalently and conformation- Institute, Yale University ally homogeneous target molecules to an exhaustive array of crystalliza- P.O. Box 208114, New Haven tion conditions is often insuf®cient to yield crystals large enough for CT 06520-8814, USA X-ray data collection. Even when large RNA crystals are obtained, they often do not diffract X-rays to resolutions that would lead to biochemi- cally informative structures. We reasoned that a well-folded RNA mol- ecule would typically present a largely undifferentiated molecular surface dominated by the phosphate backbone. During crystal nucleation and growth, this might result in neighboring molecules packing subtly out of register, leading to premature crystal growth cessation and disorder. To overcome this problem, we have developed a crystallization module consisting of a normally intramolecular RNA-RNA interaction that is recruited to make an intermolecular crystal contact. The target RNA molecule is engineered to contain this module at sites that do not affect biochemical activity. The presence of the crystallization module appears to drive crystal growth, in the course of which other, non-designed con- tacts are made. We have employed the GAAA tetraloop/tetraloop recep- tor interaction successfully to crystallize numerous group II intron domain 5-domain 6, and hepatitis delta virus (HDV) ribozyme RNA con- structs. The use of the module allows facile growth of large crystals, making it practical to screen a large number of crystal forms for favor- able diffraction properties. The method has led to group II intron domain crystals that diffract X-radiation to 3.5 AÊ resolution. # 1998 Academic Press Limited Keywords: RNA; crystallization; group II intron; HDV ribozyme; tetraloop *Corresponding author receptor Introduction 1996).Despiterecentre®nementsinmethodsto produce large quantities of covalently homo- New protein structures are now being solved geneous RNA, and the development of several daily, yet only four distinct, large RNA structures sparse matrix crystallization screens tailored for have been determined by X-ray crystallography: RNA(reviewedbyHolbrook&Kim,1997),the transferRNA(reviewedbyHolbrook&Kim, elucidation of the structures of large, functional 1997),thehammerheadribozyme(Pleyetal., RNA molecules remains a challenging endeavor 1994a;Scottetal.,1995b),theP4-P6domainofthe because of the dif®culty in obtaining large, well- TetrahymenathermophilagroupIintron(Cateetal., ordered crystals that yield X-ray diffraction data to 1996)andthe62-nucleotidefragmentIfromEscher- resolutions that allow biochemical insight. ichiacoli5SribosomalRNA(Correlletal.,1997). A critical step for macromolecular crystallization The crystallization and structure solution of short is the design of the target molecule. The designer oligonucleotide duplexes has become almost rou- endeavors to maintain biochemical activity, while tine(Bermanetal.,1996),ashasthestructuredeter- maximizing the ``crystallizability'' of the molecule mination of small RNA fragments by nuclear of interest. Historically, protein crystallographers magneticresonancespectroscopy(Varanietal., have used highly puri®ed molecules from natural sources, often performing crystallization trials with Abbreviations used: DLS, dynamic light-scattering; homologous proteins from different species. Since HDV, hepatitis delta virus; TR, tetraloop-tetraloop homologues differ primarily in their molecular sur- receptor. faces, a molecule from one source fortuitously 0022±2836/98/230621±11 $25.00/0 # 1998 Academic Press Limited 622 A General Module for RNA Crystallization might be easier to crystallize because of the par- similar RNAs shows that the tetraloop receptor/ ticular juxtaposition of functional groups of neigh- tetraloop interaction can be exploited in different boring molecules in a crystal. Limited proteolysis molecular contexts. The ease of crystallization of target molecules has been employed to remove allowed us to perform a systematic search for good ¯exible, unstructured regions, increasing confor- crystals and we report one crystal form of a mational purity, and thus, crystallizability 70-nucleotide RNA that diffracts X-rays to at least (McPherson,1982).Theadventofmolecularbio- 3.5 AÊ resolution. logical methods has made the production of mol- ecules with unnatural ends (e.g. a single domain from a multi-domain molecule) routine. Since an Results and Discussion unfortunate choice of molecular ends can result in Group II intron domains 5 and 6 as conformationally heterogeneous molecules that are crystallization targets dif®culttocrystallize(FerreÂ-D'AmareÂ&Burley, 1994),domainelucidationanddomainborderde®- Our ®rst crystallization target was an RNA nition have become important issues in the design molecule comprising domains 5 and 6 (d5 and d6) ofrecombinantcrystallizationtargets(Cohen, of the yeast mitochondrial group II self-splicing 1996). intron ai5g. Group II introns are catalytic RNAs Similar considerations apply to RNA crystalliza- found in organellar genomes of fungi and plants, tion. Since the majority of RNA targets are pre- andinsomebacteria(reviewedbyMichel&Ferat, pared not from natural sources, but by in vitro 1995).Atelevatedtemperaturesandionicstrength enzymatic or chemical synthesis, design decisions in vitro, they excise themselves from the transcript must be made from the outset. The double helix is and ligate the ¯anking exons. These introns are the basic element of RNA structure. Because of the very long, often more than 1000 nucleotides, and strength of stacking interactions in aqueous show minimal sequence conservation. Their solutions, the ends of RNA duplexes are almost sequences can, however, be arranged into a con- invariably involved in molecular contacts in crys- served pattern of six predominantly helical tals. Therefore, choice and systematic variation of domains(Micheletal.,1989;Figure1(a)).Splicing the ends of helices is as useful in RNA crystalliza- proceeds through two sequential transesteri®ca- tion as it is in DNA or DNA-protein complex crys- tions. In the ®rst, the 20 hydroxyl group of a con- tallization(e.g.Aggarwal,1990;Schultzetal.,1990; served ``bulged'' adenosine base in d6 attacks the Andersonetal.,1996).Helicalregionsoflarger 50 exon-intron junction, forming the free 50 exon RNA molecules can be predicted with some accu- and a lariat intron. In the second step, the terminal racy using phylogenetic covariation analysis or hydroxyl group of the 50 intron attacks the bound- enzymatic probing; furthermore, the solvent acces- ary of the intron and the 30 exon, releasing the sibility of helix faces can be explored chemically. In ligated exons and a lariat intron. Water competes the case of the hammerhead ribozyme, a large with the adenosine 20 hydroxyl group as the body of biochemical data guided the design of a nucleophileforthe®rsttransesteri®cation(Daniels number of crystallization targets varying in strand etal.,1996).Thishydrolyticpathwayresultsin composition and connectivity, helix length and ligated exons and a linear intron. ends(Pleyetal.,1993;Scottetal.,1995a).Knowl- The only stretch of highly conserved sequence in edge of the position of helical elements within the group II introns is the 34 nucleotide d5; the group I intron also aided in the design of minimal sequence conservation underscores its central role functional and crystallizable domains in that sys- in forming the active site. Deletion of d5 from an tem(Doudnaetal.,1993;Doudna&Cech,1995). intron abolishes activity; however, d5 can be As with proteins, variation in solvent-exposed added in trans to the rest of the intron to reconsti- regions of RNA, such as loops, can result in tutesplicing(Jarrelletal.,1988).Deletionofd6,or improvedcrystalquality(Goldenetal.,1997). of the branch-point adenosine base, results in an Here, we advance a new tool for crystallizing intron that splices using exclusively the hydrolytic RNA that differs fundamentally from well-estab- pathwayinthe®rststep(Chin&Pyle,1995).The lished ``helix engineering''. We have placed ®rst step of splicing can be reconstituted by mixing designed intermolecular, or crystal, contacts into an RNA comprising a 50 exon and d1 through d3 crystallization targets. These contacts were intro- (ExD123) with d5, d5 binding to ExD123 with a duced in the form of a bipartite crystallization dissociationconstantof800nM(Pyle&Green, module consisting of an 11-nucleotide tetraloop 1994).Theresultingreactioniskineticallyand receptor and a GAAA tetraloop, positioned to stereochemically similar to the one that takes place make exclusively intermolecular contacts, without intheintactintron(Pyle&Green,1994;Podaretal., adversely affecting biochemical activity of the tar- 1995;Michels&Pyle,1995).Thus,theisolatedd5 get molecules. Introduction of the module trans- appears to adopt a conformation similar to that formed a fragment of the active site of a group II one present in the active RNA. Examination of the intron, and a hepatitis delta virus (HDV) ribozyme, role of d5, both in binding to the rest of the intron from molecules not amenable to crystallization to and in catalysis,
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