Mechanism-based strategies for trapping and crystallizing complexes of RNA-modifying enzymes

Abstract

Posttranscriptional chemical modifications of RNA are maturation steps necessary for their correct functioning in translation during protein synthesis. Various structures of RNA-modifying enzymes complexed with RNA fragments or full-length tRNA have been obtained, mimicking several stages along the catalytic cycle such as initial RNA binding, covalent intermediate formation, or RNA-product binding. We summarize here the strategies that have been used to trap and crystallize these stable complexes. Absence of the cosubstrate transferring the chemical group leads to the Michaelis complex, whereas use of a cosubstrate analog to a ternary complex. 5-fluoro-pyrimidine-containing mini RNAs have been used as a general means to trap RNA m(5)U methyltransferase covalent complexes and RNA product/pseudouridine synthase complexes. Altogether, these structures have brought key information about enzyme/RNA recognition and highlighted the details of several catalytic steps of the reactions.