Two-step mechanism and step-arrest mutants of NAD-dependent tRNA 2'-phosphotransferase Tpt1.

Tpt1 catalyzes the transfer of an internal 2'-monophosphate moiety (2'-PO) from a "branched" 2'-PO RNA splice junction to NAD to form a "clean" 2'-OH, 3'-5' phosphodiester junction, ADP-ribose 1″-2″ cyclic phosphate, and nicotinamide. First discovered as an essential component of the tRNA splicing machinery, Tpt1 is widely distributed in nature, including in taxa that have no yeast-like RNA splicing system. Here we characterize the RslTpt1 protein from the bacterium , in which Tpt1 is encoded within a putative RNA repair gene cluster. We find that (i) expression of RslTpt1 in yeast complements a lethal Δ knockout, and (ii) purified recombinant RslTpt1 is a bona fide NAD-dependent 2'-phosphotransferase capable of completely removing an internal 2'-phosphate from synthetic RNAs. The in vivo activity of RslTpt1 is abolished by alanine substitutions for conserved amino acids Arg16, His17, Arg64, and Arg119. The R64A, R119A, and H17A mutants accumulate high levels of a 2'-phospho-ADP-ribosylated RNA reaction intermediate (2'-P-ADPR, evanescent in the wild-type RslTpt1 reaction), which is converted slowly to a 2'-OH RNA product. The R16A mutant is 300-fold slower than wild-type RslTpt1 in forming the 2'-P-ADPR intermediate. Whereas wild-type RsTpt1 rapidly converts the isolated 2'-P-ADPR intermediate to 2'-OH product in the absence of NAD, the H17A, R119A, R64A, and R16A mutant are slower by factors of 3, 33, 210, and 710, respectively. Our results identify active site constituents involved in the catalysis of step 1 and step 2 of the Tpt1 reaction pathway.