Fig. 7.

Protein-dependent and self-splicing of GsI-IIC. (a) Protein-dependent and self-splicing reactions of GsI-IIC from precursor RNAs with different 5′ exons. ³²P-labeled precursor RNAs (10 nM) were incubated with GsI-IIC RT (20 nM) for 10 min at 50 °C in reaction medium containing 5 mM Mg²⁺ (protein-dependent splicing conditons), without protein in reaction medium containing 100 mM Mg²⁺ (self-splicing conditions), no Mg²⁺ (non-splicing control), or 5 mM Mg²⁺ (5 mM; control for self-splicing under protein-dependent splicing conditions). Bands are identified to the right of the gel. The gel is split to show differently exposed top and bottom portions. (b) Time courses of the protein-dependent splicing reaction. 40 nM GsI-IIC RNA was incubated with various amounts of purified GsI-IIC RT (20 to 200 nM, color coded as indicated to the right). Samples were taken at different times, and the products were analyzed in a denaturing 4% polyacrylamide gel, which was dried and scanned with a Phosphorimager. The sets of curves at the top and bottom show disappearence of precursor RNA and appearance of intron lariat RNA, respectively. k₁ and k₂ indicate the fast and slow rate constants obtained from fitting the data to an equation with two exponentials using Prism6 (GraphPad Software).