TRNA Fusion to Streamline RNA Structure Determination: Case Studies in Probing Aminoacyl-tRNA Sensing Mechanisms by the T-Box Riboswitch

Abstract

RNAs are prone to misfolding and are often more challenging to crystallize and phase than proteins. Here, we demonstrate that tRNA fusion can streamline the crystallization and structure determination of target RNA molecules. This strategy was applied to the T-box riboswitch system to capture a dynamic interaction between the tRNA 3′-UCCA tail and the T-box antiterminator, which senses aminoacylation. We fused the T-box antiterminator domain to the tRNA anticodon arm to capture the intended interaction through crystal packing. This approach drastically improved the probability of crystallization and successful phasing. Multiple structure snapshots captured the antiterminator loop in an open conformation with some resemblance to that observed in the recent co-crystal structures of the full-length T box riboswitch–tRNA complex, which contrasts the resting, closed conformation antiterminator observed in an earlier NMR study. The anticipated tRNA acceptor–antiterminator interaction was captured in a low-resolution crystal structure. These structures combined with our previous success using prohead RNA–tRNA fusions demonstrates tRNA fusion is a powerful method in RNA structure determination.