Structure of tRNA splicing enzyme Tpt1 illuminates the mechanism of RNA 2'-PO₄ recognition and ADP-ribosylation.

Abstract

Tpt1 is an essential agent of fungal tRNA splicing that removes the 2'-PO₄ at the splice junction generated by fungal tRNA ligase. Tpt1 catalyzes a unique two-step reaction whereby the 2'-PO₄ attacks NAD⁺ to form an RNA-2'-phospho-ADP-ribosyl intermediate that undergoes transesterification to yield 2'-OH RNA and ADP-ribose-1″,2″-cyclic phosphate products. Because Tpt1 is inessential in exemplary bacterial and mammalian taxa, Tpt1 is seen as an attractive antifungal target. Here we report a 1.4 Å crystal structure of Tpt1 in a product-mimetic complex with ADP-ribose-1″-phosphate in the NAD⁺ site and pAp in the RNA site. The structure reveals how Tpt1 recognizes a 2'-PO₄ RNA splice junction and the mechanism of RNA phospho-ADP-ribosylation. This study also provides evidence that a bacterium has an endogenous phosphorylated substrate with which Tpt1 reacts.