Activity and substrate specificity of Candida, Aspergillus, and Coccidioides Tpt1: essential tRNA splicing enzymes and potential antifungal targets

Fungal Tpt1 proteins have RNA 2′-phosphotransferase activity in vitro. (A) Aliquots (5 µg) of purified recombinant AfuTpt1, CimTpt1, CalTpt1, and CauTpt1 as specified were analyzed by SDS-PAGE. The Coomassie blue-stained gel is shown. (B) Structure of the 2′-PO₄ dinucleotide in the 5′ ³²P-labeled 6-mer RNA substrate. (C–F) Reaction mixtures (10 µL) containing 100 mM Tris-HCl, pH 7.5, 2 mM DTT, 1 mM NAD⁺, 0.2 µM (2 pmol) 5′ ³²P-labeled 6-mer 2-PO₄ RNA, and CauTpt1 (C), CimTpt1 (D), CalTpt1 (E), or AfuTpt1 (F) as specified on the x-axis were incubated at 37°C for 30 min. The reaction products were analyzed by urea-PAGE. The extents of formation of the 2′-OH product are plotted as a function of input Tpt1. Each datum is the average of three independent titration experiments ±SEM.