tRNA elbow modifications affect the tRNA pseudouridine synthase TruB and the methyltransferase TrmA.

tRNAs constitute the most highly modified class of RNA. Every tRNA contains a unique set of modifications, and Ψ55, mU54, and mG46 are frequently found within the elbow of the tRNA structure. Despite the abundance of tRNA modifications, we are only beginning to understand the orchestration of modification enzymes during tRNA maturation. Here, we investigated whether pre-existing modifications impact the binding affinity or catalysis by tRNA elbow modification enzymes. Specifically, we focused on the enzymes TruB, TrmA, and TrmB which generate Ψ55, mU54, and mG46, respectively. tRNAs containing a single modification were prepared, and the binding and activity preferences of purified TrmA, TruB, and TrmB were examined in vitro. TruB preferentially binds and modifies unmodified tRNA. TrmA prefers to modify unmodified tRNA, but binds most tightly to tRNA that already contains Ψ55. In contrast, binding and modification by TrmB is insensitive to the tRNA modification status. Our results suggest that TrmA and TruB are likely to act on mostly unmodified tRNA precursors during the early stages of tRNA maturation whereas TrmB presumably acts on later tRNA intermediates that are already partially modified. In conclusion, we uncover the mechanistic basis for the preferred modification order in the tRNA elbow region.