The C-Terminal Domain of Rpb1 Functions on Other RNA Polymerase II Subunits  Hyunsuk Suh, Dane Z. Hazelbaker, Luis M. Soares, Stephen Buratowski  Molecular Cell  Volume 51, Issue 6, Pages 850-858 (September 2013) DOI: 10.1016/j.molcel.2013.08.015 Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 Transferring the C-Terminal Domain of RPB1 to Different Locations of RNA Polymerase II Complex (A) Crystal structure of S. pombe RNApII (PDB ID code 3H0G; Spåhr et al., 2009). Rpb1 linker residues 1,451–1,496 just upstream of the CTD repeats (Rpb1 C′) are highlighted in red, Rpb4 in violet, Rpb6 in cyan, and Rpb9 in orange. RNApII is shown from three perspectives, with “Front” designating the view from the downstream template DNA. (B) Scheme of fusion constructs. RPB1ΔCTD lacks 25 heptad repeats of the C-terminal domain in RPB1. Three versions of CTD with differing lengths of the Rpb1 linker were fused to the C terminus of Rpb4, Rpb6, or Rpb9 as indicated. (C) Rpb4, Rpb6, or Rpb9 fused to CTD complements deletion of the corresponding genes. Plasmids carrying the indicated RPBn constructs were transformed into strains lacking the genomic copy of RPB4, RPB6, or RPB9 either directly (RPB4) or by plasmid shuffling (RPB6 and RPB9). For rpb4Δ complementation testing, growth was measured at 25°C (permissive temperature) or 30°C (nonpermissive) on synthetic complete media (SC) plates. For rpb6Δ or rpb9Δ, the covering wild-type plasmid was removed by 5-FOA selection. Three-fold serial dilutions of cultures were spotted onto plates and incubated as labeled. (D) The Rpb1 CTD is functional on Rpb4 or Rpb6, but not Rpb9. Strains carrying deletions of the genomic RPB1 and indicated RPBn genes were used for plasmid shuffling of the indicated plasmids. Left panel shows growth on SC plates before shuffling, while the right panel shows the result of 5-FOA selection. The total number of CTDs per RNApII molecule is listed on the right. See Figure S1 for additional related results. Molecular Cell 2013 51, 850-858DOI: (10.1016/j.molcel.2013.08.015) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 CTD-Fusion Proteins Are Stably Expressed and Phosphorylated In Vivo (A) Wild-type (WT, YSB2898), Rpb4-sL-CTD (4C, YSB2899), and Rpb6-sL-CTD (6C, YSB2900) strains were created by plasmid shuffling as in Figure 1. Whole-cell extracts were separated by SDS-PAGE, and blots were probed with the indicated antibodies. Protein size estimates (kDa) to the right are based on protein size markers. Rpb3 level (bottom strip) was used as a loading control for total RNApII. See Figure S2 for additional related results. (B) RNApII was immunoprecipitated using anti-Rpb3 antibody and probed with the indicated antibodies (listed to left). The first three lanes show 5% of the input whole-cell lysates, the next three lanes show the immunoprecipitated fractions, and the last three lanes show the unbound supernatant. Asterisks (∗) indicate immunoglobulin or nonspecific crossreactive bands. Molecular Cell 2013 51, 850-858DOI: (10.1016/j.molcel.2013.08.015) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 ChIP Analysis of RNApII, CTD Phosphorylations, and CTD-Recruited Factors in CTD Transfer Strains The (A) PMA1, (B) ADH1, (C) snR13, and (D) snR33 genes were analyzed for crosslinking of the indicated proteins in wild-type (WT), Rpb4-sL-CTD (4C), and Rpb6-sL-CTD (6C) strains. Three RNApII subunits (Rpb3, Rpb4, and Rpb6); CTD phosphorylated at Ser5, Ser7, or Ser2; and CTD-binding proteins Ceg1, Nrd1, and Rna15 were tested. For each panel, the schematic at top shows the PCR primer regions (numbered boxes) along the gene. Graphs underneath show the average of quantified results from three independent ChIP experiments, with the y axis representing fold enrichment over a nontranscribed background signal and error bars showing standard error. All experiments used chromatin from the same strain, except for Nrd1 (∗), which was examined in strains carrying TAP-tagged Nrd1. Representative gel images are shown in Figure S3. Molecular Cell 2013 51, 850-858DOI: (10.1016/j.molcel.2013.08.015) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 ChIP Analysis of RNApI, RNApIII, and CTD Phosphorylations on Rpb6-sL-CTD Rpc40, a common subunit of RNApI and RNApIII, was TAP tagged in Rpb6 (WT) or Rpb6-sL-CTD (6C) strain backgrounds. Crosslinking was analyzed at (A) RNApI-transcribed 35S rDNA and (B) RNApIII-transcribed SUP56 (Leucyl-tRNA) gene regions. Precipitation was with IgG beads for RPC40-TAP, or antibodies against Rpb6, Ser5-P (3E8), Ser7-P (4E12), or Ser2-P (H5). For each panel, the schematic at top shows the PCR primer regions (numbered boxes) along the gene. Graphs underneath show the average of quantified results from three independent ChIP experiments, with the y axis representing fold enrichment over a nontranscribed background signal, and error bars showing standard error. Representative gel images are shown in Figure S4. Molecular Cell 2013 51, 850-858DOI: (10.1016/j.molcel.2013.08.015) Copyright © 2013 Elsevier Inc. Terms and Conditions