Structure and two-metal mechanism of fungal tRNA ligase.

ABSTRACT: Fungal tRNA ligase (Trl1) is an essential enzyme that repairs RNA breaks with 2',3'-cyclic-PO4 and 5'-OH ends inflicted during tRNA splicing and non-canonical mRNA splicing in the fungal unfolded protein response. Trl1 is composed of C-terminal cyclic phosphodiesterase (CPD) and central GTP-dependent polynucleotide kinase (KIN) domains that heal the broken ends to generate the 3'-OH,2'-PO4 and 5'-PO4 termini required for sealing by an N-terminal ATP-dependent ligase domain (LIG). Here we report crystal structures of the Trl1-LIG domain from Chaetomium thermophilum at two discrete steps along the reaction pathway: the covalent LIG-(lysyl-N?)-AMP•Mn2+ intermediate and a LIG•ATP•(Mn2+)2 Michaelis complex. The structures highlight a two-metal mechanism whereby a penta-hydrated metal complex stabilizes the transition state of the ATP ? phosphate and a second metal bridges the ? and ? phosphates to help orient the pyrophosphate leaving group. A LIG-bound sulfate anion is a plausible mimetic of the essential RNA terminal 2'-PO4. Trl1-LIG has a distinctive C-terminal domain that instates fungal Trl1 as the founder of an Rnl6 clade of ATP-dependent RNA ligase. We discuss how the Trl1-LIG structure rationalizes the large body of in vivo structure-function data for Saccharomyces cerevisiae Trl1.

SUBMITTER: Banerjee A 

PROVIDER: S-EPMC6379707 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature