Cysteine-Pair Cross-Linking Assay

For Cys triplet reporter (CTR) cross-linking assays (Figure 6B,E and F), ECs and PECs were assembled on purified DNA and RNA scaffolds specified in the figure legend and as described previously (Kang et al., 2018a (link)). Briefly, 10 μM RNA, 12 μM template DNA, and 15 μM non-template DNA (Supplementary file 1) were annealed in RB. To assemble complexes, scaffold (2 μM) was mixed with limiting CTR RNAP (1 μM; CTR RNAP: β′1045iC 258iC, β843C) in 50 mM Tris-HCl, pH 7.9, 20 mM NaCl, 10 mM MgCl₂, 0.1 mM EDTA, 5% glycerol, and 2.5 μg of acetylated bovine serum albumin/mL, and added to mixtures of cystamine and DTT to generate redox potentials that ranged from −0.314 to −0.424. Complexes were incubated for 60 min at room temperature and then were quenched with the addition of iodoacetamide to 15 mM. The formation of cysteine-pair cross-links was then evaluated by non-reducing SDS-PAGE (4%–15% gradient Phastgel; GE Healthcare) as described previously (Nayak et al., 2013 (link)). Gels were stained with Coomassie Blue and imaged with a CCD camera. The fraction cross-linked was quantified with ImageJ software. The experimental error was determined as the standard deviation of measurements from three or more independent replicates.