Ja. Doudna et al., CRYSTALLIZATION OF RIBOZYMES AND SMALL RNA MOTIFS BY A SPARSE-MATRIX APPROACH, Proceedings of the National Academy of Sciences of the United Statesof America, 90(16), 1993, pp. 7829-7833
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.