Fig. 1.

TbCe1 is a cytoplasmic capping enzyme that possesses 5′-monophosphate RNA kinase and guanylyltransferase activities. (A) Localization of PTP-tagged TbCgm1 and TbCe1. Procyclic trypanosomes expressing either TbCgm1–PTP (Top) or TbCe1–PTP (Bottom) were fixed and permeabilized, and the PTP–tag was detected by immunofluorescence. The kinetoplast and nuclear DNA were counterstained with DAPI. A typical image is shown. (B) TbCe1 specifically phosphorylates pRNA. Reaction mixtures (10 µL) containing 50 mM Tris⋅HCl (pH 8), 100 μM [γ-³²P] ATP, 1 mM DTT, 0.5 mM MgCl₂, and either 2 pmol of 21-mer pppRNA, 21-mer pRNA, 24-mer _OHRNA₂₄, or _OHRNA₂₄ treated with polynucleotide kinase (pRNA₂₄), as indicated, were incubated with 20, 100, or 500 ng of TbCe1 (proceeding from Left to Right within each titration series). A control reaction lacking enzyme (−) is shown in the lane indicated. (C) RNA capping activity. Reaction mixtures (10 µL) containing 50 mM Tris⋅HCl (pH 8.5), 1 mM DTT, 0.5 mM MgCl₂, 20 µM [α-³²P] GTP, 100 nM of pRNA, with TbCe1 and ATP concentration as specified. Position of the capped-labeled 21-mer RNA is indicated. Radiolabeled phosphate is indicated in red.