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. 2021 Sep 28;12:5610. doi: 10.1038/s41467-021-25870-3

Fig. 1. Assembly and catalysis of recombinant human TSEN.

Fig. 1

a Bar diagrams of TSEN subunits (TSEN15, green; TSEN34, orange; TSEN2, red; TSEN54, blue) and CLP1 (brown) depicting positions of PCH mutations, predicted nuclease domains of TSEN2 and TSEN34, and the RNA kinase domain of CLP1. The total amino acids of each protein are indicated. b SDS-PAGE of purified recombinant TSEN and TSEN/CLP1 complexes visualized by InstantBlue staining. Protein identities and size markers are shown. c Native mass spectrum of tetrameric TSEN complex from an aqueous ammonium acetate solution. Charge states of the predominant TSEN2–15–34–54 heterotetramer (blue circles), Heat Shock Protein (HSP) 70 (gray circles), the heterodimer TSEN15–34 (red circles), and TSEN15 (green circles) are indicated. d Pre-tRNA cleavage assay using tetrameric TSEN complex with Saccharomyces cerevisiae (S.c.) pre-tRNAPheGAA and mature tRNAPheGAA. Input samples and cleavage products were separated via urea-PAGE and visualized by Toluidine blue. RNA denominations are given on the right. e Pre-tRNA cleavage assay with TSEN heterodimers and S.c. pre-tRNAPheGAA. SDS-PAGE of the indicated heterodimers and the reconstituted TSEN tetramer is shown on the left (InstantBlue stain), urea-PAGE of the cleavage products on the right (Toluidine blue stain). Gels are representative of three independent experiments. Source data for b, d, and e, are provided as Source Data files.