IMPORTANT PARAMETERS FOR RNA CRYSTALLOGENESIS. Joseph D. Ng, Brice Felden, Gérard Keith, Philippe Dumas, Philippe Brion, Anne Dietrich-Théobald, Catherine Florentz and Richard Giegé. UPR 9002 Structure des Macromolécules Biologiques et Mécanismes de Reconnaissance, Institut de Biologie Moléculaire et Cellulaire du CNRS,15 rue René Descartes, 67084 Strasbourg,Cedex, France

We present two model systems showing important parameters that may facilitate RNA crystal growth. First, we exemplify the importance of important RNA crystal growth features with the crystallization of a synthetic RNA minisubstrate derived from the brome mosaic virus (BMV) tRNA-like structure that was devised and synthesized to form a small RNA circle of 24 nucleotides (24cRNA). When the 24cRNA is hybridized with a 10 base complementary single-stranded RNA terminating with a CCA 3'-terminus, the RNA duplex becomes an efficient substrate for yeast histidyl-tRNA synthetase (1). This small RNA complex is arranged in a pseudoknot-like fold and is referred to as a resected pseudoknot. Using established RNA purification procedures sufficient in producing molecules for molecular biology or biochemical activity studies, small amounts of contamination, mainly of low but also of high molecular weights are still not removed. We show that these contaminants are obtained during the course of purification from electrophoretic gel matrixes or buffers revealed by NMR analysis. The elimination of these trace contaminants was critical for the successful crystallization of this molecule. Moreover, temperature was also determined to be important in crystallizing the small RNA circle such that crystals can only be obtained at 30^oC or higher.

Secondly, certain additives affect positively RNA crystal growth. This is illustrated by examining a series of cationic additives which were used to study the crystallization of tRNAs. These reagents were observed to decrease nucleation and increase crystal size.

Reference: (1) Felden, B. (1994). Thése de l'Université Louis Pateur, Strasbourg.