Transitions between the steps of forward and reverse splicing of group IIC introns

Effects of IEP and π mutations on Ta.it.I1 branched splicing. (A) Superimposed structures of a cryo-EM IIB RNP structure (PDB 6MEC, magenta) and an active group IIC IEP (PDB 6AR1, teal). (B) Alignment of amino acids predicted to interact with DVI. Basic amino acids (blue) underlined were mutated to E in this study (Ta.it.) or previously (T.el.; Haack et al. 2019). (C) RT assay of purified WT and mutant IEPs. (D) Pull-down assay of His tagged IEPs against Ni-agarose resin to assess RNP formation. (E) WT and mutant IEPs assayed with WT or π mutant Ta.it.I1 RNA in 10 mM MgCl₂ and 0.5 M NH₄Cl at 55°C. (F) Time course of WT and π mutant Ta.it.I1 RNA spliced with WT IEP in 10 mM MgCl₂ and 0.5 M NH₄Cl at 55°C.