Fig. 4.

EAF1-dependent stimulation of ELL elongation activity requires both the ELL N terminus and elongation activation domain. (A) Diagram of wild-type ELL and ELL mutants used in the experiments of B and C, showing the positions of the elongation activation (elongation), lysine-rich (K rich), and occludin-like domains. (B) Interaction of EAF1 and truncation mutant ELL proteins in Sf21 insect cells. Sf21 cells were infected with recombinant baculoviruses encoding the EAF1 protein with an N-terminal Myc tag, and the truncated ELL proteins were indicated with an N-terminal FLAG tag. Forty-eight hours after infection, cells were lysed as described in Materials and Methods and centrifuged at 20,000 × g for 30 min at 4°C. The resulting supernatants were subjected to immunoprecipitation with anti-FLAG agarose, and bound proteins were eluted from anti-FLAG beads with a 150 μg/ml FLAG peptide. Proteins present in anti-FLAG agarose eluates were fractionated by SDS/PAGE and visualized by Western blotting with anti-FLAG (red) or anti-myc (green) antibodies. (C) Two (lanes 2 and 3) or 8 pmol (lanes 5-11) of the indicated factors were assayed for their ability to stimulate synthesis of 135-nt transcripts from the T-less cassette of oligo(dC)-tailed template pAd-GR220. Transcription was initiated by the addition of 50 μM ATP, 50 μM GTP, and 10 μCi [α-³²P]CTP (400 mCi/mmol), and reactions were incubated for 15 min.