EAF1 stimulates ELL elongation activity in the absence of additional transcription factors. Oligo(dC)-tailed template transcription reactions were initiated by the addition of 50 μM ATP, 50 μM GTP, 2 μM CTP, and 10 μCi [α-³²P]CTP (400 mCi/mmol). After a 30-min incubation, 135-nt transcripts were chased into longer products in the presence of 2 μM UTP and 100 μM CTP, with or without 1 pmol ELL or 0.5 pmol EAF1 as indicated. Reaction products were analyzed as described for promoter-specific transcription assays. The lane marked M shows ΦX174 HaeIII restriction fragments used as size markers. To the right of the gel is diagrammed the oligo(dC)-tailed template used in these assays, showing the position of the oligo(dC)-tail (C_n)C_OH and the T rich stretches in the nascent transcript.

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.

Transcript elongation activity of ELL is stimulated by EAF1. (A) Diagram of EcoRI to NdeI fragment of pDN-AdML, which contains AdML promoter sequences from -50 to +10 inserted into the polylinker of pUC18 and extends 254 nt downstream of the transcriptional start site (+1). The sequence of the nascent transcript from +1to +15 is shown. (B) Diagram of the reaction protocol used in the experiment of C. GTFs, general transcription factors TATA-box binding protein, TFIIB, TFIIE, TFIIF, and TFIIF; A, ATP; G, GTP; C, CTP; U, UTP; ³²PC, [α-³²P]CTP (C) ELL and EAF1, both with N-terminal FLAG tags, were expressed in Sf21 insect cells and purified by anti-FLAG affinity chromatography as described in Materials and Methods. Promoter-specific transcription reactions were performed as described in Materials and Methods. Transcription was initiated by the addition of 50 μM ATP, 50 μM UTP, and 10 μCi [α-³²P]CTP. After 30 min (lane 1), reactions were chased with 100 μM CTP and 1 μM GTP in the presence or absence of 1 pmol ELL and 0.5 pmol EAF1 as indicated (lanes 2-9). A14 indicates the position of the nascent transcript that is synthesized in the presence of ATP, CTP, and UTP and stalled at the A residue at +14. The black arrowhead indicates the position of the full-length, 254-nt runoff transcript. Numbers on the left indicate the position of ΦX174 HaeIII restriction fragments used as size markers.

Cochromatography of ELL stimulatory activity with EAF1. (A) Approximately 15 μg of FLAG-EAF1, which had been purified by anti-FLAG agarose chromatography, was resolved on a 0.6 ml TSK DEAE-NPR HPLC column as described under Materials and Methods. The indicated fractions were resolved on a 12% SDS polyacrylamide gel and visualized by silver staining (Ag stain). (B) One-microliter aliquots of the indicated fractions were assayed for their ability to stimulate synthesis of 135-nt transcripts from the T-less cassette of oligo(dC)-tailed template pAd-GR220 in the presence of ELL (lanes 5 to 14). Controls (lanes 1 to 4) consist of reactions with pol II alone (lane 1) or supplemented with 1 pmol of the indicated proteins. Transcription was initiated by the addition of 50 μM ATP, 50 μM GTP, 2 μM CTP, and 10 μCi [α-³²P]CTP (400 mCi/mmol) and stopped after a 5-min incubation.

Both EAF1 and EAF2 stimulate ELL elongation activity. Two picomoles of EAF1 or EAF2 was added to reactions with or without 1 pmol of either ELL or ELL2 as indicated. Transcription was initiated by addition of 50 μM ATP, 50 μM GTP, 2 μM CTP, and 10 μCi [α-³²P]CTP and incubated for 5 min.