CRYSTALLIZATION OF RIBOZYMES AND SMALL RNA MOTIFS BY A SPARSE-MATRIX APPROACH

The three-dimensional structures of RNA enzymes form catalytic centers that include specific substrate binding sites. High-resolution determ ination of these and other RNA structures is essential for a detailed understanding of the function of RNA in biological systems. The crysta l structures of only a few RNA molecules are currently known. These in clude tRNAs, which were produced in vivo and contained modified bases, and short oligonucleotide duplexes lacking tertiary interactions. Her e we report that a number of different RNA molecules of 4-50 kDa, all synthesized in vitro, have been crystallized. A highly successful meth od for the growth of RNA crystals based on previously reported conditi ons for tRNA crystallization is presented. This method is rapid and ec onomical, typically requiring 1.1 mg of RNA to set up an experiment an d 2 weeks to complete the observations. Using this technique, we have obtained crystals of 8 of 10 different RNA molecules tested, ranging i n size from a dodecamer duplex to a 208-nucleotide catalytic intron. S everal of these crystal forms diffract to high resolution; in one case , we have collected a 2.8-angstrom native data set for a 160-nucleotid e domain of the group I self-splicing intron from Tetrahymena thermoph ila. The solution of these RNA structures should reveal aspects of ter tiary structure that relate to RNA function and catalytic mechanisms.