Structures of RNA phosphotransferase Tpt1 reveal distinct binding modes for an RNA 2'-PO splice junction versus a 5'-PO mononucleotide.

Tpt1 is a widely distributed enzyme that removes an internal RNA 2'-phosphate by transfer to NAD, via a two-step reaction in which: (i) the RNA 2'-PO attacks NAD to form an RNA-2'-phospho-(ADP-ribose) intermediate and expel nicotinamide; and (ii) the ADP-ribose O2″ attacks the RNA 2'-phosphodiester to form 2'-OH RNA and ADP-ribose-1″,2″-cyclic phosphate products. Tpt1 can also execute a single-step ADP-ribosyltransferase reaction at a 5'-monophosphate nucleic acid terminus that installs a 5'-phospho-ADP-ribose cap structure. Here we present crystal structures of Tpt1 bound to an RNA containing an internal 2'-PO mark (the substrate for the canonical Tpt1 pathway) and in a complex with 5'-AMP. We find that Tpt1 has distinct binding modes, whereby the RNA 2'-PO and the AMP 5'-PO are engaged by the same set of active site amino acids, but the 2'-PO nucleoside and the 5'-nucleoside occupy different sites on the enzyme.