Kinetic and structural insights into the requirement of fungal tRNA ligase for a 2'-phosphate end.

Fungal RNA ligase (LIG) is an essential tRNA splicing enzyme that joins 3'-OH,2'-PO and 5'-PO RNA ends to form a 2'-PO,3'-5' phosphodiester splice junction. Sealing entails three divalent cation-dependent adenylate transfer steps. First, LIG reacts with ATP to form a covalent ligase-(lysyl-Nζ)-AMP intermediate and displace pyrophosphate. Second, LIG transfers AMP to the 5'-PO RNA terminus to form an RNA-adenylate intermediate (AppRNA). Third, LIG directs the attack of an RNA 3'-OH on AppRNA to form the splice junction and displace AMP. A defining feature of fungal LIG vis-à-vis canonical polynucleotide ligases is the requirement for a 2'-PO to synthesize a 3'-5' phosphodiester bond. Fungal LIG consists of an N-terminal adenylyltransferase domain and a unique C-terminal domain. The C-domain of LIG (CthLIG) engages a sulfate anion thought to be a mimetic of the terminal 2'-PO Here, we interrogated the contributions of the C-domain and the conserved sulfate ligands (His227, Arg334, Arg337) to ligation of a pRNAp substrate. We find that the C-domain is essential for end-joining but dispensable for ligase adenylylation. Mutations H227A, R334A, and R337A slowed the rate of step 2 RNA adenylation by 420-fold, 120-fold, and 60-fold, respectively, vis-à-vis wild-type CthLIG. An R334A-R337A double-mutation slowed step 2 by 580-fold. These results fortify the case for the strictly conserved His-Arg-Arg triad as the enforcer of the 2'-PO end-specificity of fungal tRNA ligases and as a target for small molecule interdiction of fungal tRNA splicing.