Structural basis for Tpt1-catalyzed 2'-PO&lt;sub&gt;4&lt;/sub&gt; transfer from RNA and NADP(H) to NAD<sup/>.

Abstract

Tpt1 is an essential agent of fungal and plant tRNA splicing that removes an internal RNA 2'-phosphate generated by tRNA ligase. Tpt1 also removes the 2'-phosphouridine mark installed by Ark1 kinase in the V-loop of archaeal tRNAs. Tpt1 performs a two-step reaction in which the 2'-PO₄ attacks NAD⁺ to form an RNA-2'-phospho-(ADP-ribose) intermediate, and transesterification of the ADP-ribose O2″ to the RNA 2'-phosphodiester yields 2'-OH RNA and ADP-ribose-1″,2″-cyclic phosphate. Here, we present structures of archaeal Tpt1 enzymes, captured as product complexes with ADP-ribose-1″-PO₄, ADP-ribose-2″-PO₄, and 2'-OH RNA, and as substrate complexes with 2',5'-ADP and NAD⁺, that illuminate 2'-PO₄ junction recognition and catalysis. We show that archaeal Tpt1 enzymes can use the 2'-PO₄-containing metabolites NADP⁺ and NADPH as substrates for 2'-PO₄ transfer to NAD⁺. A role in 2'-phospho-NADP(H) dynamics provides a rationale for the prevalence of Tpt1 in taxa that lack a capacity for internal RNA 2'-phosphorylation.