Structures of RNA phosphotransferase Tpt1 reveal distinct binding modes for an RNA 2′-PO₄ splice junction versus a 5′-PO₄ mononucleotide

Tpt1 reaction mechanism and structure of PhoTpt1 in complex with 2′-PO₄ RNA. (A) The Tpt1 reaction pathway comprises the two chemical steps shown in which: (i) the RNA 2′-PO₄ reacts with NAD⁺ to expel nicotinamide and form a 2′-phospho-ADP-ribosylated RNA intermediate; and (ii) transesterification of the ADP-ribose 2″-OH to the RNA 2′-PO₄ displaces the RNA 2′-OH and generates ADP-ribose-1″,2″-cyclic phosphate. (B) Tertiary structure of PhoTpt1, depicted as a cartoon model with magenta β-strands, cyan α-helices, and blue 3₁₀ helices. The ApA^2′PpA substrate (stick model with gray carbons and yellow phosphorus atoms) occupies the RNA lobe. (C) Stereo view of the ApA^2′PpA substrate overlaid with a simulated annealing omit density map (blue mesh contoured at 1 σ), highlighting its atomic interactions with amino acids in the N-terminal α1 helix of PhoTpt1 (depicted as stick models with beige carbons). Hydrogen bonds are denoted by black dashed lines. Van der Waals contacts are depicted as beige dashed lines.