Fig. 5. NF1 protein is involved in multiple-round transcription from the VA1 template. (A) The NF1-binding site in the terminator region of the VA1 gene contributes to the multiple-round transcription by RNA polymerase III. In vitro transcription reactions were performed with HeLa nuclear extract using super-coiled (SC) (lanes 1–4 and 9) or linear (L) (lanes 5–8 and 10–13) templates under conditions for the multiple- (lanes 1–8) or single-round (lanes 9–13) transcription. VA1 templates with the wild-type terminator (lanes 1, 4, 5, 9 and 10), a mutated NF1-binding site (lanes 2, 6 and 11), a mutated terminator (lanes 7 and 12) or a mutated NF1-binding site and a mutated terminator (lanes 3, 8 and 13) are indicated as WT, MT(NF1), MT(T) and MT(NF1/T), respectively. The ratio of the signals obtained with the mutant templates (MT) relative to that obtained with the wild-type template (WT) is indicated at the bottom as percentage activity. The origin of the weak bands (lanes 5, 10 and 11) just above the RT products is unknown, but they may be generated from the vector sequence during transcription. (B) NF1 is required for transcription from the VA1 template. Western blot analysis was performed with anti-CTF1 antibodies to determine the nature and levels of NF1 proteins in the HeLa nuclear extract depleted with pre-immune (PI, lane 1) or immune (I, lane 2) anti-CTF1 antibodies. In vitro transcription reactions with the VA1 template were performed with HeLa nuclear extracts immunodepleted with either pre-immune (lane 3) or immune (lanes 4–9) antibodies against human CTF1. Reaction mixtures were complemented with either 2 (lane 5) or 6 µl (lane 6) of the purified factor or with 5 (lane 7), 10 (lane 8) or 20 ng (lane 9) of recombinant CTF1.