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. 1999 Nov 23;96(24):13720–13725. doi: 10.1073/pnas.96.24.13720

Figure 5.

Figure 5

Effects of rhTRF2 and flag⋅hTRF2–TFIIA on transcription by RNA polymerase III. (A) Transcription of the 7SK gene. System C contained 1.5 and 10 μl of fractions cEDF 1 M and cEDF 0.2 M, respectively, prepared as described in Materials and Methods and 24 ng of a rhTFIIIB component (TFIIIB150) related to Saccharomyces cerevisiae TFIIIB90 (M.T., Z.W., M. Ito, K. H. Seifart, and R.G.R., unpublished data). Individual lanes were supplemented with the following transcription factors. Lane 1: none; lanes 2 and 3: 20 and 40 ng of rhTBP; lanes 4–6 and 9–11: 20, 40, and 80 ng of rhTRF2, respectively; lanes 7 and 8 and 12 and 13: 20 and 40 ng of flag-tagged human TRF2, complexed with TFIIA (approximately 80 and 160 ng of protein in total); lanes 9–13 contained, in addition, 40 ng of rhTBP. (B) Transcription of the VAI gene. System D contained 0.5 μl of core TFIIIC purified from cell line Cβ (37), 0.1 μl of core RNA polymerase III purified from cell line BN51 (38), 150 ng of rhPC4 (21), 6 ng of rhTFIIIB150 (M.T., Z.W., M. Ito, K. H. Seifart, and R.G.R., unpublished data), and 6 ng of rhTFIIIB90 (4). Other additions were as follows: 5 ng of rhTBP in lanes 1–5; 6 ng of rhTFIIA in lanes 2, 5, 7, and 10; 6 ng of rhTRF2 in lanes 3, 5, 8, and 10; and 24 ng of purified flag⋅hTRF2–TFIIA complex (6 ng flag⋅hTRF2) in lanes 4 and 9.