Crystals by design: a strategy for crystallization of a ribozyme derived from the Tetrahymena group I intron.
Recently, the 2.8 A crystal structure of one domain of the self-splicing Tetrahymena group I intron was reported. Although it revealed much about RNA tertiary interactions, it contained only half of the active site. We have now designed a series of larger molecules that contain about 70% of the intron and all of the catalytic core. These RNAs were efficient in cleavage of a substrate RNA, consisting of the approximately 100 nucleotides from the 5' end of the intron, at a site corresponding to the 5' splice site. A sparse matrix was designed specifically for large RNAs and used to screen for preliminary crystallization conditions. Of the six RNAs initially tested, five were crystallized in this initial trial. Two of these crystals were further examined. The first diffracted X-rays to only approximately 16 A resolution, even when the crystal were very large. The second diffracted as high as 3.5 A, but the crystals were twinned and therefore unusable for structural studies. Site-specific mutagenesis was performed on the latter RNA to disrupt interactions that might have been responsible for the twinning. One of these mutant RNAs produced large, single, diffraction-quality crystals. The crystals belong to the tetragonal space group P42212 and have large unit cell dimensions, a=b=178 A and c=199 A. Thus, by variation of both sequence elements and crystallization conditions, crystals of a 247 nucleotide catalytic RNA were obtained.
Golden
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Howard Hughes Medical Institute, University of Colorado, Boulder, CO 80309, USA.
Podell
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Gooding
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Cech
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D000818:Animals; D003460:Crystallization; D018360:Crystallography, X-Ray; D007438:Introns; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013768:Tetrahymena / Q000201:enzymology*
D016337:RNA, Catalytic