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. 1999 Feb;19(2):1605–1615. doi: 10.1128/mcb.19.2.1605

FIG. 2.

FIG. 2

Ectopic TFIIIC can relieve chromatin-mediated repression of RNA polymerase III transcription in nuclear extracts. (A) Analysis of immunopurified TFIIIC by SDS-PAGE (8% gel) and silver staining. Lane 1, molecular weight markers; lane 2, control immunoprecipitation; lane 3, immunopurified purified TFIIIC. The marked bands in lane 3 represent the TFIIIC2 polypeptides. (B) Transcription from chromatin and naked DNA templates. Freshly assembled chromatin and naked DNA (20 fmol) were preincubated with or without TFIIIC (260 fmol [100 ng]) at 30°C for 20 min before nuclear extract (NE) addition, as indicated. The amount of endogenous TFIIIC in the added nuclear extract (1 μl) was 1.2 fmol. Lanes 1 and 2, chromatin transcription; lanes 3 and 4, DNA transcription. (C) Effect of TFIIIC addition before (pre) or after (post) chromatin assembly. Either 100 or 200 ng of TFIIIC was added before (lanes 2 and 3) or after assembly (lanes 4 and 5) chromatin assembly. Assembled templates were subjected to in vitro transcription as described above. (D) Ectopic TFIIIC does not enhance transcription at limiting concentrations of DNA template. Transcription of 2 ng (lane 1 and 2), 4 ng (lanes 3 and 4), 6 ng (lanes 5 and 6), and 50 ng (lanes 7 and 8) of DNA was performed with HeLa nuclear extract in the absence (lanes 1, 3, 5, and 7) or presence (lanes 2, 4, 6, and 8) of 100 ng of TFIIIC. (E) Histones and NAP1 do not affect transcription from the DNA template. Histones and NAP1 were incubated in chromatin assembly buffer for 15 min and then added to in vitro transcription reactions with or without TFIIIC. Transcription reaction mixtures contained nuclear extract only (lane 1), nuclear extract and 100 ng TFIIIC (lane 2), nuclear extract and histones plus NAP1 (lane 3), or nuclear extract, 100 ng TFIIIC, and histones plus NAP1 (lane 4). (F) An equimolar amount of chromatin template does not affect transcription from the DNA template. Transcription reaction mixtures with nuclear extract contained 20 fmol of DNA (lane 1), 20 fmol of DNA, and 20 fmol of chromatin (chro) (lane 2), 20 fmol of chromatin (lane 3), or 20 fmol of chromatin with 100 ng of purified TFIIIC.