Fig. 3

TFIIH-dependent activation of co-transcriptional capping in artificial ternary complexes assembled on DNA:RNA scaffolds. a Scheme for assembly of artificial ternary elongation complexes on DNA:RNA scaffolds. See text and “Methods” for details. b Representative gel image showing step-wise increase of RNA length following addition of appropriate combinations of rNTPs. 20-nt synthetic RNA in artificial ternary complexes was extended to position +23 with 5 µM ATP and 10 µCi α-³²P-UTP (first lane), washed and walked to position +25 with 20 µM CTP and ATP (second lane), and finally washed and walked to position +29 with 20 µM ATP and GTP (last lane). Sequence on left denotes RNA sequence from +21 (bottom) to +29 (top). c 23mers in artificial ternary complexes were incubated with buffer or 300 ng of purified TFIIH for 10 min, washed, and incubated for 1, 2, or 4 min with 50 µM GTP and the indicated amounts of capping enzyme. Graph shows mean and range of data from two independent reactions