Volume 44, Issue 3, Pages 397-409 (November 2011) Sub1 and RPA Associate with RNA Polymerase II at Different Stages of Transcription  Timothy W. Sikorski, Scott B. Ficarro, John Holik, TaeSoo Kim, Oliver J. Rando, Jarrod A. Marto, Stephen Buratowski  Molecular Cell  Volume 44, Issue 3, Pages 397-409 (November 2011) DOI: 10.1016/j.molcel.2011.09.013 Copyright © 2011 Elsevier Inc. Terms and Conditions

Molecular Cell 2011 44, 397-409DOI: (10.1016/j.molcel.2011.09.013) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 Sub1 and RPA Are Enriched on Promoter Sequences In Vitro (A and B) Representative spectra from low mass iTRAQ reporter region of a (A) Sub1 peptide and (B) Rfa1 peptide. Enrichment is measured by taking the ratios of the areas under each of the peaks. Figure S1C shows spectra for Rfa3 and a nonenriched peptide. (C) The preferential interaction between Sub1 and the HIS4 promoter template was confirmed by immobilized template binding of nuclear extracts containing a TAP-tagged Sub1 on the HIS4 and no promoter templates. Bound proteins were analyzed by immunoblotting using antibodies against Rpb3 (a subunit of RNApII), TBP, Rfa1, Pol2 (a subunit of DNA polymerase), and the TAP tag (for Sub1). Molecular Cell 2011 44, 397-409DOI: (10.1016/j.molcel.2011.09.013) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 Sub1 Is Found at Promoters (A) Schematic representations of the YEF3 and PMA1 genes used in ChIP experiments are shown at top. The box shows the open-reading frame and arrows indicate transcription start site. Bars below the genes represent the positions of the ChIP PCR products (see Table S4 for primer sequences). ChIP was performed using a TAP-tagged Sub1 strain and representative gels are shown in middle panels. DNA coprecipitated with IgG-agarose (to precipitate TAP-tagged Sub1) was analyzed by multiplex PCR using primers shown in the schematic. The TEL PCR product is an internal background control from a nontranscribed region. The input control is used to normalize for PCR amplification efficiency of each primer pair. Quantitations of experiments are shown at bottom. The values shown represent the averages and standard errors (bars) from three independent experiments. (B) ChIP analysis at the YEF3 gene was performed for TBP (left) and Sub1-TAP (right) in wild-type TBP and TBP(Y94C) strains. Quantitation of triplicate experiments is as in (A). (C) ChIP of the Rpb3 subunit of RNApII at the PMA1 and YEF3 genes was performed in strains with SUB1 or sub1Δ backgrounds. Quantitation of triplicate experiments is as in (A). Molecular Cell 2011 44, 397-409DOI: (10.1016/j.molcel.2011.09.013) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 Sub1 Interacts Genetically with TFIIE and TFIIH (A) Growth of TFA1, SUB1(YSB290); TFA1, sub1Δ (YSB2530); tfa1(212-482Δ), SUB1 (YSB318); tfa1(212-482Δ), sub1Δ (YSB2532); tfa1(C124S), SUB1 (YSB335); and tfa1(C124S), sub1Δ (YSB2534) strains was assayed by 10-fold serial dilutions and incubation at 23°C, 30°C, or 37°C for 3 days. (B) Growth of SSL1, SUB1 (YF28); ssl1-1, SUB1 (YF29); SSL1, sub1Δ (YSB2578); and ssl1-1, sub1Δ (YSB2579) strains was assayed as in (A). Molecular Cell 2011 44, 397-409DOI: (10.1016/j.molcel.2011.09.013) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 Sub1 Localizes Near the Transcription Bubble (A) The top panel shows a representation of Sub1 protein domain structure. Below are the positions of the FeBABE labels in each probe (numbered brackets) shown above the HIS4 promoter sequence. The TATA element and the predicted transcription bubble region are indicated. (B) Cleavage fragments of TAP-tagged Sub1 were resolved by gel electrophoresis, blotted, and visualized by probing for the TAP protein A domain at the Sub1 carboxyl terminus. All 12 probes were conjugated to FeBABE and used in PIC formation/hydroxyl radical probing as described in Miller and Hahn, 2006. Negative controls include probe 3 reactions lacking either FeBABE or H2O2. Molecular Cell 2011 44, 397-409DOI: (10.1016/j.molcel.2011.09.013) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 5 RPA Associates with Active Genes (A) ChIP was performed using an antibody against the Rfa1 subunit of RPA. Coprecipitated DNA was analyzed across the PMA1, YEF3, and PYK1 genes as described in Figure 2. The values shown represent the averages and standard errors (bars) from three independent experiments. (B) ChIP was performed using strains expressing TAP-tagged Dpb2 or Pol2, (components of DNA Polymerase [II] ε), or Orc1 (component of the replication origin recognition complex). DNA coprecipitated with the TAP-tagged proteins was analyzed by PCR for regions across the YEF3 gene. (C) Cells were grown in YP-Glucose or YP-Galactose to an OD ∼0.5, and Rfa1 ChIP was performed as above except testing for regions across the GAL10 gene. Quantitation of triplicate experiments is as in (A). (D) Strains with the indicated genotypes were spotted in 3-fold dilutions on synthetic complete (SC) + 5-Fluoroorotic acid to remove an RFA1-URA3 covering plasmid. Plates were incubated for 6 days at 30°C. (E) The indicated isogenic strains were spotted in 3-fold dilutions on SC plates lacking uracil (3 days growth shown) or SC-URA plates containing MPA (4 days). (F) ChIP of the Rpb3 subunit of RNApII at the YEF3 gene was performed in strains with wild-type Rfa1 (RFA1) or expressing the rfa1-t124 allele grown in galactose. Quantitation of triplicate experiments is as in (A). Molecular Cell 2011 44, 397-409DOI: (10.1016/j.molcel.2011.09.013) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 6 RPA Associates with Transcribed Regions of the Genome (A) Comparison of Rfa1 and Rpb3 (RNApII) occupancy at coding sequences throughout the genome. ChIP was performed as in Figure 5, except that DNA was labeled and used to probe genome oligonucleotide arrays. Scatter plot represents all probes plotted for the log2 ratio between the immunoprecipitated DNA and a total DNA control. tRNA probes are marked in red. (B) Comparison of Rfa1 and Rpb3 (RNApII) occupancy at probes classified as “mRNA midcoding” sequences. Molecular Cell 2011 44, 397-409DOI: (10.1016/j.molcel.2011.09.013) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 7 RPA Crosslinks to Transcription Start Sites in the Absence of Sub1 Activity (A) ChIP analysis at the PYK1 gene was performed using antibody against Rfa1 in SUB1 and sub1Δ strains. The values shown represent the averages and standard errors (bars) from three independent experiments. (B) Left panel: Scatter plot of all probes on the genomic arrays, comparing Rfa1 occupancy in SUB1 and sub1Δ cells. Right panel: Probes that are >1.5-fold increased in sub1Δ cells are shown. Loci annotated as “transcription start sites” (TSS) are marked in red. (C) Coincidence test for increase in Rfa1 occupancy in sub1Δ cells for each locus category. P value indicates probability a particular locus category is represented in the increased category by chance. N/A refers to categories that had no loci increased in sub1Δ cells. (D) ChIP analysis for Rfa1 at the PYK1 gene was performed using chromatin samples from sub1Δ or sub1Δ cells carrying plasmid-borne SUB1 or sub1(Y66A) alleles. Quantitation of triplicate experiments is as in (A). (E) ChIP for Sub1-HA was performed in a strain bearing a tagged version of wild-type Sub1 or the Y66A mutant, and PCR analysis of the precipitated DNA was carried out on the PYK1 gene. Quantitation of triplicate experiments is as in (A). Molecular Cell 2011 44, 397-409DOI: (10.1016/j.molcel.2011.09.013) Copyright © 2011 Elsevier Inc. Terms and Conditions