Fig. 4. The VA1 terminator-binding factor belongs to the NF1 protein family and interacts directly with TFIIIC2 subunits. (A) DNA sequence of two terminator (T1 and T2) regions of the VA1 gene. The protected regions observed in the footprint assays and the consensus binding site for the NF1 protein family are indicated. (B) The interaction of CTF1 with the terminator region of the VA1 gene is dependent on theNF1 consensus sequence. The recombinant CTF1 shifted the VA1 terminator-containing probe in a dosage-dependent manner (lanes 2and 3) and the mutation of the NF1 consensus sequence in the probe diminished the shift (lanes 5 and 6). One (lanes 2 and 5) or 5 (lanes 3 and 6) ng of CTF1 expressed in bacteria were used in gel shift assay. (C) NF1 family proteins are present in holo RNA polymerase III, holo TFIIIC and TFIIIC1 fractions. HeLa cell nuclear extract (2.5 µl) (lane 1), holo RNA polymerase III (lane 2), core RNA polymerase III (lane 3), holo TFIIIC (lane 4), core TFIIIC purified under the salt concentrations of 300 (lane 5) or 800 (lane 6) mM KCl and TFIIIC1 fraction (lane 7) were resolved by 10% SDS–PAGE and subjected to immunoblot analysis with antibodies against the N-terminus of CTF1. Arrows indicate the polypeptides recognized by the antibodies and positions of the molecular size makers (kDa) are indicated on the left. (D) CTF1 interacts directly with TFIIIC220 and TFIIIC110. M2 agarose beads alone (lanes 2, 5, 8, 11 and 14) or M2 agarose beads containing 100 ng of the FLAG-tagged CTF1 expressed in Sf21 cells were subjected to individual pull-down assays with the ³⁵S-labeled subunits of TFIIIC2 indicated at the top of lanes. Lanes 1, 4, 7, 10 and 13 are 20% of input.