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Local RNA structural changes induced by crystallization are revealed by SHAPE
Local RNA structural changes induced by crystallization are revealed by SHAPE.
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We present a simple approach to locate sites that undergo conformational changes upon crystallization by comparative structural mapping of the same RNA in three different environments. As a proof of principle, we probed the readily crystallized P4-P6DeltaC209 domain from the Tetrahymena thermophila group I intron in a native solution, in a solution mimicking the crystallization drop, and in crystals. We chose the selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry, which monitors the flexibility and the conformation of each nucleotide. First, SHAPE successfully revealed the structural changes that occur during the crystallization process. Specifically, 64% of the nucleotides implicated in packing contacts and present in the portion of the molecule analyzed were identified. Second, reactivity differences for some of these nucleotides were already observed in the crystallization solution, suggesting that the crystallization buffer locked down a particular structure that was favorable to crystal formation. Third, the probing of a known structure extends our understanding of the structural basis for the SHAPE reaction by suggesting that reactivity is enhanced by a C2'-endo sugar pucker. Furthermore, by identifying local conformational changes of the RNA that take place during crystallization, SHAPE could be combined with the in vitro selection of stable mutants to rationalize the design of RNA candidates for crystallization.
Vicens Q, Gooding AR, Laederach A, Cech TR
RNA (New York, N.Y.)
2007-04-01 00:00
13
4
536-48
Acylation,Animals,Base Pairing,Base Sequence,Buffers,Crystallization,Crystallography, X-Ray,Hydrogen Bonding,Introns,Models, Molecular,Molecular Sequence Data,Nucleic Acid Conformation,RNA,RNA, Catalytic,RNA, Protozoan,Solutions,Tetrahymena thermophila,Buffers,RNA, Catalytic,RNA, Protozoan,Solutions,RNA
Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, Univeristy of Colorado, Boulder, CO 80309-0215, USA quentinvicenscoloradoedu
RNA
NIGMS P01 GM66275, NIGMS U54 GM072970
1355-8382
10.1261/rna.400207
rna.400207
206
True
17299128
