Extended Data Fig. 1: Biochemical investigation of group II intron branching.

The reactions are conducted under a condition that strongly favours intron branching (50 mM NH₄-HEPES pH 7.5, 500 mM NH₄Cl and 30 mM MgCl₂). a. A denaturing 5% PAGE gel showing the time course of E.r. intron self-splicing. The intron precursor RNA is radiolabeled with ³²P uridine and the gel is imaged by autoradiography. Hydrolysis is the predominant splicing pathway, giving only linear intron. b. A quantitative plot of the E.r. intron self-splicing time course. Individual in vitro splicing time course with one independent isolated intron RNA and maturase sample is shown. c. A denaturing 5% PAGE gel showing the time course of E.r. intron splicing in the presence of the maturase protein (4-molar excess to the precursor RNA). With the facilitation of its cognate maturase protein, the major splicing pathway of E.r. intron is switched to branching, yielding dominantly lariat intron. Individual in vitro splicing time course with one independent isolated intron RNA and maturase sample is shown. d. A quantitative plot of the maturase-mediated E.r. intron splicing time course. The apparent precursor depletion rate increases from 0.03 min⁻¹ in the case of self-splicing to 0.16 min⁻¹ with maturase assistance. Gel filtration chromatogram of (e) the pre-1F and pre-2F and (f) the post-2F RNP sample preparation. e. The two peaks correspond to aggregates, co-eluted pre-1F and pre-2F RNP sample respectively. f. The two peaks arise from the post-2F RNP sample and the spliced exons respectively. A₂₆₀ and A₂₈₀ traces are shown in red and blue respectively.