Analysis of cross-links in complexes H and A with [APB+9B]-pre-mRNA. (A) Gradient profiles of complex H (incubation with [APB+9B]-pre-mRNA for 10 min on ice) and complex A (10 min at 30°C) after centrifugation in a 1.5 mL S55-S rotor. Complex H sediments at around 12 S, complex A at around 20 S, and complex B, which appears as a minor product, at around 40 S. (B) Nondenaturing gel analysis of gradient fractions 1–7 from the 30°C incubation. A total of 10 μL from each fraction was applied to a 2% nondenaturing agarose gel. The complexes are identified at right. (C) Photoaffinity cross-linking at 312 nm after gradient centrifugation. Aliquots from fractions 1, 3, 5, and 7 of B were cross-linked, and parallel aliquots from fractions 3, 5, and 7 were subjected to identical procedures, but without UV irradiation. Radioactive proteins were then separated on a 10% SDS–polyacrylamide gel. (Lane 1) Products of the 30°C incubation cross-linked without prior centrifugation (input, “i”); (lanes 2–5) fractions 1, 3, 5, and 7 from B after cross-linking; (lanes 6–8) as in lanes 3–5, but in a mock cross-linking reaction without UV light applied. (M) Prestained molecular-weight markers (Bio-Rad) are indicated at left. The asterisk indicates the RNase T1-digested non-cross-linked RNA.
