Abstract: |
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 '-PO4 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 '-PO4 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 '-PO4 and the AMP 5 '-PO4 are engaged by the same set of active site amino acids, but the 2 '-PO4 nucleoside and the 5 '-nucleoside occupy different sites on the enzyme. |