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A Metazoan/Plant-like Capping Enzyme and Cap Modified Nucleotides in the Unicellular Eukaryote Trichomonas vaginalis

Figure 5

RNA capping activity of recombinant TvCE.

Capping activity was detected in a two-step reaction, monitoring both TPase and GTase activities, using [α-32P]GTP and a 352 nt RNA substrate. Wild-type TvCE (WT), a TPase-inactive mutant (C126S) and a GTase-inactive mutant (R526S) were tested in the two-step reaction using various combinations as indicated in the black filled squares (top panel). A negative control reaction using the RNA substrate dephosphorylated by alkaline phosphatase prior to capping was included as indicated (AP). The final product was split equally and loaded on two polyacrylamide gels that were subsequently analyzed by autoradiography (middle panel) or ethidium bromide staining (bottom panel). Arrows denote molecular weights of radiolabeled RNA markers (Century Marker Plus, Ambion) and the RNA substrate.

Figure 5

doi: https://doi.org/10.1371/journal.ppat.1000999.g005