Characterization of tRNA splicing enzymes RNA ligase and tRNA 2′-phosphotransferase from the pathogenic fungi Mucorales

Canonical pathway of fungal tRNA splicing and structural organization of the trifunctional fungal tRNA ligase Trl1. (A) Intron removal by tRNA splicing endonuclease leaves 2′,3′-cyclic phosphate and 5′-OH ends on the broken tRNA halves. The tRNA exons are then joined by Trl1—a trifunctional tRNA ligase. Trl1 catalyzes two end-healing reactions, performed by a 5′-OH polynucleotide kinase domain and a polynucleotide 2′,3′-cyclic phosphodiesterase (CPD) domain, to generate the 5′-PO₄ and 3′-OH,2′-PO₄ termini required for sealing by an ATP-dependent RNA ligase domain. The 2′-PO₄ at the resulting splice junction is removed by the NAD⁺-dependent RNA 2′-phosphotransferase enzyme Tpt1. (B) Fungal Trl1 consists of N-terminal ligase, central kinase, and C-terminal CPD catalytic modules. The structures of the ligase (pdb 6N0T), kinase (pdb 6U03), and CPD (pdb 6U05) domains from the indicated fungi are shown in complexes with active site ligands.