Figure 7.

Mss116p-promoted splicing of group II introns aI5γ and bI1. Splicing time courses for aI5γ (left) and bI1 (right) were done by incubating 20 nM ³²P-labeled RNA substrate with 100 nM Mss116p protein and 1 mM ATP in reaction medium containing 100 mM KCl and 8 mM MgCl₂ at 30°C. Splicing products were analyzed in a denaturing 4% polyacrylamide gel, which was dried and quantified with a phosphorimager. The bottom segments of the gels are darker exposures to show E1 and E2, which result form SER. Control lanes show RNA substrate incubated for 120 min with Mss116p plus 1 mM AMP-PNP (+AMP-PNP) or without Mss116p (-Mss116p). The additional lanes (far right) show precursor RNAs incubated with wild-type (WT) Mss116p and mutant Mss116p-K158E under the same conditions for 120 min. The plots beneath the gels show disappearance of precursor RNA and appearance of products as a function of time, with the data fit to a single exponential. The table at the bottom summarizes k_obs values, with the numbers in parentheses indicating the concentration of RNA (nM) reacted or produced after 120 min. The I-Lin band may contain a mixture of linear intron and broken lariat RNA. Abbreviations: E1, 5′ exon; E2, 3′ exon; E1–E2, ligated exons; I-Lar, intron lariat; I-Lin, linear intron; P, precursor RNA.