Fig. 5: T. brucei CBP66 is the dsRNA-binding subunit of TbCBC.
From: Structural basis of Spliced Leader RNA recognition by the Trypanosoma brucei cap-binding complex
A EMSA of TbCBC-tetramer binding to the SL RNA exons with different 5’ chemistry. B Size exclusion chromatography of the engineered TbCBP66* (CBP30(G141-N220)-CBP66 fusion), a representative curve of more than 3 experiments is shown. C EMSA probing the interaction of TbCBP66* with different uncapped RNAs: the 39 nt SL RNA exon (same ligand as in Fig. 3F, G), a 15 nt single-stranded RNA (A1-U15 of the SL RNA), and the 25 nt SL RNA hairpin loop comprising A14-G39 of the SL RNA. EMSAs in (A) and (C) were run in triplicate reactions, and the intensity of PAGE gel bands was normalized in relation to the protein-free RNA control band; A Hill model was used to fit the binding curve. D The Alphafold2 model of TbCBP66 indicates three domains with exposed positive charges on all domains. A surface representation of the Alphafold2 model and the Predicted aligned error (PAE) plot indicating the domain position confidence are shown. E Fluorescence polarization (FP) assay probing the binding of the 39 nt SL RNA exon to TbCBP66* single point mutants. The assay was run in triplicate and fit with a nonlinear sigmoidal fit model. See also Supplementary Fig. S12. Source data are provided as a Source Data file.
