A Minimal RNA Polymerase III Transcription System from Human Cells Reveals Positive and Negative Regulatory Roles for CK2  Ping Hu, Si Wu, Nouria Hernandez  Molecular Cell  Volume 12, Issue 3, Pages 699-709 (September 2003) DOI: 10.1016/j.molcel.2003.08.011

Figure 1 Composition of the Pol III, SNAPc, TBP, Brf2, and Bdp1 Preparations The factors shown are indicated above the lanes. Lane 1 is reprinted from Figure 1 in Hu et al. (2002). Lanes 3 and 4 are from the same gel. In lane 1, asterisks mark pol III subunits and dots mark abundant, copurifying proteins. In lane 2, the arrowheads indicate SNAP190, SNAP50, SNAP45, SNAP43, and SNAP19, and in lanes 3–5, Brf2, TBP, and Bdp1, respectively. The faint bands in lanes 3 and 4 probably correspond to E. coli contaminants. Molecular Cell 2003 12, 699-709DOI: (10.1016/j.molcel.2003.08.011)

Figure 2 The Tagged Pol III Complex Can Direct U6 Transcription When Complemented with Recombinant SNAPc, TBP, Brf2, and Bdp1 (A) A minimal in vitro U6 transcription system. In lanes 1–4, the indicated amounts of HeLa whole-cell extracts were used for in vitro U6 transcription. In lanes 5–8, 1, 2, 4, and 8 μl of pol III complex were complemented with recombinant TBP (30 ng), Brf2 (50 ng), Bdp1 (100 ng), and SNAPc (400 ng). Lanes 9–14 show either a complete reconstituted reaction (lane 9) or reactions missing the components indicated above the lanes (lanes 10–14). U6 5′, correctly initiated U6 RNA; IC (internal control), RNA added to the transcription reaction to control for recovery. (B) RPC1 contents in whole-cell extracts and the pol III complex. The amounts of whole-cell extract or purified tagged pol III complex indicated above the lanes (in μl) were probed with an anti-RPC1 antibody. (C) SNAPc, TBP, Brf2, and Bdp1 are not limiting in the whole-cell extract. In lanes 2–4, increasing amounts of SNAPc (160, 320, and 800 ng), TBP (15, 45, and 90 ng), Brf2 (30, 90, and 150 ng), Bdp1 (80, 160, and 400 ng), or SNAPc, TBP, Brf2, and Bdp1 (labeled “all”, the same amounts of each factor were added in lanes 2–4 as when added individually) were added to the whole-cell extract. (D) U6 template and riboprobe used to map the 5′ and 3′ ends of the transcripts. The riboprobe extends from the NdeI site 70 nt upstream of the transcription start site to the T3 promoter about 100 nt past the U6 3′ end. Correctly initiated and terminated RNA protects the probe over 181 nucleotides. (E) The RNAs synthesized in the reconstituted system are correctly terminated. An RNase T1 protection was performed with RNA synthesized either in the whole-cell extract (lane 1) or in the reconstituted system (lane 2) from the template shown in (D). Molecular Cell 2003 12, 699-709DOI: (10.1016/j.molcel.2003.08.011)

Figure 3 The Reconstituted System Directs Several Rounds of Transcription (A) A 60 min preincubation allows for maximum transcription preinitiation complex formation. 4 μl of tagged pol III complex was preincubated at 30°C with transcription factors as in Figure 2A and 80 ng of U6 DNA template in the absence of nucleotides. After 30, 60, and 90 min, the reactions were either stopped (lanes 1, 3, and 5) or complemented with all four nucleotides and incubated for another 3 min (lanes 2, 4, and 6). Transcription was not detectable in a 3 min reaction without preincubation (data not shown). (B) Accumulation of correctly initiated RNA in the presence of sarkosyl. Reactions were assembled and preincubated for 60 min as in (A), then complemented with 0.075% sarkosyl and all four nucleotides and incubated for the times indicated above the lanes. (C) Accumulation of correctly initiated RNA in the absence of sarkosyl. Reactions were assembled and processed as in (B) except without sarkosyl. (D) The U6 signals obtained in (B) and (C) were quantitated with a Fuji PhosphorImager, corrected for background, and normalized for the internal control signal. Relative transcription efficiencies are indicated. Molecular Cell 2003 12, 699-709DOI: (10.1016/j.molcel.2003.08.011)

Figure 4 Further Purification of the Tagged Pol III Complex (A) The purified pol III complex does not contain detectable topoisomerase 1, PC4, La, and NF-1. 40, 80, and 120 μg of whole cell extract and 260 ng of tagged pol III complex were analyzed by immunoblot for the proteins indicated on the right of each panel. Note that in a typical reconstituted transcription reaction, 50 ng of tagged pol III complex is used. (B) Purification scheme of the tagged pol III complex. (C) Each mini Q fraction was complemented with recombinant SNAPc, TBP, Brf2, and Bdp1 and tested for U6 transcription activity. The peak of transcription activity in fractions 9–12 eluted between 340 and 460 mM KCl. (D) Immunoblots showing the distributions of various proteins (indicated on the right of each panel) in the mini Q fractions. The last four panels are from a separate experiment in which pol III peaked in fraction 10. Molecular Cell 2003 12, 699-709DOI: (10.1016/j.molcel.2003.08.011)

Figure 5 CK2 Is Found Associated with the U6 Promoter Region In Vivo (A) Crosslinked and sonicated chromatin from dividing HeLa cells was immunoprecipitated with antibodies against the SNAP190 subunit of SNAPc (antibody CS696; lanes 2 and 3), the corresponding preimmune antibodies (lanes 4 and 5), no antibodies (lanes 6 and 7), and antibodies against TFIIB (CS396; lanes 12 and 13), Brf2 (CS1228; lanes 16 and 17), CK2α (lanes 20 and 21), and CK2β (lanes 24 and 25). In lanes 10, 11, 14, 15, 18, 19, 22, and 23, no chromatin was added (mock immunoprecipitations). In each case, the immunoprecipitated material was analyzed by PCR with test (T) primers specific for the U6 or U1 promoters, and control (C) primers hybridizing to regions 4 and 7 kb upstream of the U6 or U1 snRNA genes, respectively. In lanes 8 and 9, the chromatin starting material was analyzed directly by PCR. (B) Chromatin was prepared as in (A) and used for immunoprecipitation with either no antibodies (lanes 5 and 6), anti-SNAP190 antibodies (lanes 1, 2, and 7–22), or preimmune antibodies (lanes 3 and 4). The bound material was then either analyzed directly (lanes 1–6) or eluted with the SNAP190 peptide against which the anti-SNAP190 antibody was raised and used for a second immunoprecipitation with antibodies against the factors indicated above the lanes. The lanes labeled “mock” are as in (A). The material was analyzed by PCR for the presence of U6 sequences as in (A). Molecular Cell 2003 12, 699-709DOI: (10.1016/j.molcel.2003.08.011)

Figure 6 CK2 Activity Is Required for U6 Transcription in the Reconstituted System (A) Inhibitors of CK2 debilitate U6 transcription in the reconstituted system. A reconstituted U6 transcription reaction was supplemented with either buffer (lanes 1 and 6), or 6 and 12 mM 2,3-diphosphoglycerate (DPG; lanes 2 and 3) or 40 and 50 μM 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002; lanes 4 and 5) just before addition of the nucleotides. Lanes 1–3 and 4–6 are from different gels. (B) An excess of CK2 substrate debilitates U6 transcription in the reconstituted system. The transcription reaction was supplemented with either buffer (lane 1), or a CK2 phosphoacceptor peptide (RRREEETEEE) at 180 and 60 μM (lanes 2 and 3) or the same peptide at 150 μM and 0.4 and 1 unit of recombinant CK2α (lanes 4 and 5), or a control peptide (PKA phosphoacceptor peptide, LRRASLG) at 150 and 60 μM (lanes 6 and 7), or 0.4 and 1 unit of recombinant CK2α (lanes 8 and 9) just before addition of the nucleotides. Molecular Cell 2003 12, 699-709DOI: (10.1016/j.molcel.2003.08.011)

Figure 7 CK2 Can Both Activate and Inhibit Pol III Transcription by Phosphorylating Different Targets (A) CK2 activity is not required after assembly of the transcription initiation complex. Reactions were preincubated with CK2 and ATP for 30 min, then complemented with 80 μM LY294002, either 0.015% (lanes 1–6) or 0.075% (lanes 7–12) sarkosyl, and the remaining nucleotides, and incubated for the times indicated above the lanes. (B) CK2 phosphorylation of the pol III complex is required for transcription. Lane 1 shows a standard reconstitution reaction. In lanes 2–9, the factors labeled “I” above the lanes (either the pol III complex, or SNAPc, or a mixture containing TBP, Brf2, and Bdp1) were incubated separately first with 60 μM of LY294002 and then with 5 units of CK2 α2β2 and 0.5 mM ATP, and the factors labeled “CK2” were incubated first with 5 units of CK2 α2β2 and 0.5 mM ATP and then with 60 μM of LY294002. The factors were then used for in vitro transcriptions. (C) The experiment was as in (B) except that the source of phosphate during the incubations with CK2 was GTP instead of ATP. Molecular Cell 2003 12, 699-709DOI: (10.1016/j.molcel.2003.08.011)

Figure 8 A Minimal Transcription System that Directs Several Rounds of Correctly Initiated and Terminated Transcription from the Human U6 Promoter SNAPc with its subunits SNAP190, SNAP50, SNAP45, SNAP43, and SNAP19 is bound to the proximal sequence element (PSE) and TBP is bound to the TATA box. SNAPc and TBP, and TBP and Brf2, are shown contacting each other because they bind cooperatively to DNA. CK2 phosphorylation of the pol III complex is required for transcription, whereas phosphorylation of the TBP-Brf2-Bdp1 factors is inhibitory. Molecular Cell 2003 12, 699-709DOI: (10.1016/j.molcel.2003.08.011)