A GENERAL MODULE FOR RNA CRYSTALLIZATION

Crystallization of RNA molecules other than simple oligonucleotide dup lexes remains a challenging step in structure determination by X-ray c rystallography. Subjecting biochemically, covalently and conformationa lly homogeneous target molecules to an exhaustive array of crystalliza tion conditions is often insufficient to yield crystals large enough f or X-ray data collection. Even when large RNA crystals are obtained, t hey often do not diffract X-rays to resolutions that would lead to bio chemically informative structures. We reasoned that a well-folded RNA molecule 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 di sorder. To overcome this problem, we have developed a crystallization module consisting of a normally intramolecular RNA-RNA interaction tha t is recruited to make an intermolecular crystal contact. The target R NA molecule is engineered to contain this module at sites that do not affect biochemical activity. The presence of the crystallization modul e appears to drive crystal growth, in the course of which other, non-d esigned contacts are made. We have employed the GAAA tetraloop/tetralo op receptor interaction successfully to crystallize numerous group II intron domain 5-domain 6, and hepatitis delta virus (HDV) ribozyme RNA constructs. The use of the module allows facile growth of large cryst als, making it practical to screen a large number of crystal forms for favorable diffraction properties. The method has led to group II intr on domain crystals that diffract X-radiation to 3.5 Angstrom resolutio n. (C) 1998 Academic Press Limited.