DNA Topoisomerase I and PC4 Can Interact with Human TFIIIC to Promote Both Accurate Termination and Transcription Reinitiation by RNA Polymerase III  Zhengxin Wang, Robert G Roeder  Molecular Cell  Volume 1, Issue 5, Pages 749-757 (April 1998) DOI: 10.1016/S1097-2765(00)80074-X

Figure 1 Structural and Functional Analyses of the Human Holo TFIIIC (A) SDS–PAGE analysis of the sucrose gradient–purified human TFIIIC. 250 fmol of sucrose gradient–purified TFIIIC (fraction 7) was separated on SDS–8% polyacrylamide gels and stained with silver. The five subunits of TFIIIC2 and the four associated polypeptides (marked with asterisks) that specifically cosedimented with TFIIIC2 subunits are indicated in kDa. Other unmarked polypeptides (notably those around 55 kDa) did not copurify. (B) The holo TFIIIC contains two functional components. Transcription assays with the VA1 template contained 2.4 fmol of the immunopurified TFIIIB and 100 fmol of immunopurified RNA polymerase III/TDF with no addition (lane 1), 50 fmol of holo TFIIIC (lane 2), 50 fmol of the sucrose gradient–purified TFIIIC (fraction 7, SG#7) (lane 3), 1 μl of sucrose gradient fraction 14 (SG#14) (lane 4), 50 fmol of SG#7 plus 1 μl of SG#14 (lane 5) or 50 fmol of SG#7 plus 2 μl of SG#14 (lane 6). (C) Two components of holo TFIIIC are jointly required for transcription initiation by RNA polymerase III. Primer extension assays were performed following transcription of the VA1 template with the same amounts of the indicated factors in the same reconstituted system employed in (B). Molecular Cell 1998 1, 749-757DOI: (10.1016/S1097-2765(00)80074-X)

Figure 2 Coactivators Topo I and PC4 Are Found in the Holo TFIIIC Complex (A) Topoisomerase activity in holo TFIIIC versus TFIIIC. The supercoiled pH2D template (Wang and Roeder 1997) was incubated with 65 (lane 3), 130 (lane 4), or 260 (lane 5) fmol of the immunoisolated holo TFIIIC complex and with 130 (lane 6) or 260 (lane 7) fmol of the sucrose gradient–purified TFIIIC. In lane 2, 1 μg of the supercoiled pH2D template was incubated in the absence of factors. Lane 1, standard DNA markers. Arrows S and R indicate supercoiled and open, circular forms of pH2D DNA, respectively. (B) Immunoblot analysis of immunoisolated holo TFIIIC. 30 μg of HeLa cell nuclear extract (lane 1), 520 fmol of sucrose gradient–purified TFIIIC (lanes 2 and 4), and 520 fmol of holo TFIIIC (lanes 3 and 5) were analyzed by immunoblot with anti–topo I (lanes 1–3) and anti-PC4 (lanes 4–5) antibodies. The bands corresponding to topo I and PC4 are indicated. (C) SDS–PAGE analysis of purified recombinant human topo I. Coomassie blue–stained polyacrylamide gels contained 2 μg of purified wild-type (lane 2) or Y723F topo I (lane 3). Lane 1, standard size markers (in kDa). (D) Analysis of wild-type versus mutant topo I enzymatic activity. Supercoiled pH2D DNA was incubated with 0.1 (lanes 3 and 7), 1 (lanes 4 and 8), 10 (lanes 5 and 9), or 100 (lanes 6 and 10) ng of the purified recombinant wild-type topo I (lanes 7–10) or Y723F topo I (lanes 3–6). In lane 2, 1 μg of the supercoiled pH2D template was incubated in the absence of factors. Lane 1, standard DNA markers. Molecular Cell 1998 1, 749-757DOI: (10.1016/S1097-2765(00)80074-X)

Figure 3 Identification of Topo I and PC4 as Functional Components of the Human Holo TFIIIC Complex Transcription assays contained the VA1 template, 2.4 fmol of highly purified TFIIIB, and 100 fmol of immunopurified RNA polymerase III/TDF. Other additions, indicated at the top, included: none (lanes 1 and 8), 50 fmol sucrose gradient–purified TFIIIC (lanes 3, 5, 7, 9, and 11–16), 50 fmol of holo TFIIIC (lane 2), 1 μl of sucrose gradient fraction 14 (lanes 4, 5, and 9), ∼2 ng (20 fmol) of homogeneous native topo I (lanes 6 and 7), 50 ng of recombinant topo I (lanes 10 and 11), and 12.5 (lane 13), 25 (lane 14), 50 (lane 15), or 100 (lane 16) ng of recombinant PC4. Molecular Cell 1998 1, 749-757DOI: (10.1016/S1097-2765(00)80074-X)

Figure 4 Topo I and PC4 Extend TFIIIC Interactions to the Downstream Promoter Regions Footprinting analyses were performed on the lower strand of the tRNA template with 260 fmol of holo TFIIIC, 260 fmol of sucrose gradient–purified TFIIIC, and various amounts of recombinant topo I and PC4 as indicated. Lanes 1 contained Maxam-Gilbert G+A reactions and lanes 2 the naked template. Reactions in (A) employed holo TFIIIC (lane 3), TFIIIC (lane 4), 100 ng of the wild-type (lane 5) or mutant Y/F (lane 8) topo I alone, TFIIIC plus 50 (lane 6) or 100 (lane 7) ng of the wild-type topo I, and TFIIIC plus 25 (lane 9), 50 (lane 10), or 100 (lane 11) ng of the mutant Y723F topo I. Reactions in (B) contained holo TFIIIC (lane 3) or TFIIIC (lane 4) alone and 30 (lane 5), 60 (lane 6), or 120 (lane 7) ng of the recombinant PC4 alone (lanes 5–7) or together (lanes 8–10) with TFIIIC. Molecular Cell 1998 1, 749-757DOI: (10.1016/S1097-2765(00)80074-X)

Figure 5 Functional Assays of PC4 and Topo I (A) PC4 promotes multiple-round transcription by RNA polymerase III. Transcription assays with the VA1 template were performed in the absence (lanes 1 and 2) or in the presence (lanes 3 and 4) of 0.05% sarkosyl in the reconstituted system containing 10 fmol of TFIIIB, 50 fmol of TFIIIC, and 100 fmol of RNA polymerase III/TDF in the presence (lanes 2 and 4) or in the absence (lanes 1 and 3) of 100 ng of recombinant PC4. In each assay, factors were incubated with the template for 60 min prior to sarkosyl (lanes 3 and 4) or buffer (lanes 1 and 2) and nucleotide addition and a subsequent 60 min incubation. The autoradiographic exposure time for the single-round transcription assay (lanes 3 and 4) was about 20-fold longer than that for the multiple-round transcription assay (lanes 1 and 2). (B) Diagram of the C-tailed VA1 template and derived transcripts. (C) Holo TFIIIC facilitates accurate termination by RNA polymerase III on the tailed VA1 template. Assays contained 20 fmol of C-tailed VA1 template and either 24 μg of nuclear extract that had been depleted with the preimmune serum (lane 4) or anti-TFIIIB90 immune serum (lane 5) or 24 fmol of immunopurified RNA polymerase III (lanes 1–3) in the absence (lane 1) or presence of 260 (lane 2) or 520 (lane 3) fmol of holo TFIIIC. Transcripts terminated at the first termination site (T1), the second termination site (T2), or the end of the template (RT) are indicated. (D) PC4 in conjunction with TFIIIC facilitates accurate termination by RNA polymerase III on the tailed VA1 template. Assays contained 20 fmol of C-tailed VA1 template and 24 fmol of immunopurified RNA polymerase III (lanes 1–7) in the absence of additional factors (lane 1) or in the presence of 520 fmol of holo TFIIIC (lane 2), 520 fmol of TFIIIC (lane 3), 80 ng of the recombinant PC4 (lane 7), or 520 fmol of TFIIIC plus 80 (lane 4), 100 (lane 5), or 120 (lane 6) ng of recombinant PC4. (E) Differential sensitivity of transcripts to RNase H. Labeled transcripts synthesized by RNA polymerase III from the tailed template (in the absence of other factors) were treated with RNase H (lane 3). As an additional control, an aliquot of the reaction mixture was treated at 100°C for 3 min prior to the addition of RNase H (lane 2). Molecular Cell 1998 1, 749-757DOI: (10.1016/S1097-2765(00)80074-X)