Using tRNA Scaffold to Assist RNA Crystallization

Recent studies have solidified RNA’s regulatory and catalytic roles in all life forms. Understanding such functions necessarily requires high-resolution understanding of the molecular structure of RNA. Whereas proteins tend to fold into a globular structure and gain most of the folding energy from tertiary interactions, RNAs behave the opposite. Their tertiary structure tends to be irregular and porous, and they gain the majority of their folding free energy from secondary structure formation. These properties lead to higher conformational dynamics in RNA structure. As a result, structure determination proves more difficult for RNA using X-ray crystallography and other structural biology tools. Despite the painstaking effort to obtain large quantities of chemically pure RNA molecules, many still fail to crystallize due to the presence of conformational impurity. To overcome the challenge, we developed a new method to crystallize the RNA of interest as a tRNA chimera. In most cases, tRNA fusion significantly increased the conformational purity of our RNA target, improved the success rate of obtaining RNA crystals, and made the subsequent structure determination process much easier. Here in this chapter we describe our protocol to design, stabilize, express, and purify an RNA target as a tRNA chimera. While this method continues a series of work utilizing well-behaving macromolecules/motifs as “crystallization tags” (Ke and Wolberger. Protein Sci 12:306–312, 2003; Ferre-D’Amare and Doudna. J Mol Biol 295:541–556, 2000; Koldobskaya et al . Nat Struct Mol Biol 18:100–106, 2011; Ferre-D’Amare et al. J Mol Biol 279:621–631, 1998), it was inspired by the work of Ponchon and Dardel to utilize tRNA scaffold to express, stabilize, and purify RNA of interest in vivo (Ponchon and Dardel. Nat Methods 4:571-576, 2007). The “tRNA scaffold,” where the target RNA is inserted into a normal tRNA, replacing the anticodon sequence, can effectively help the RNA fold, express in various sources and even assist crystallization and phase determination. This approach applies to any generic RNA whose 5′ and 3′ ends join and form a helix.