When listing more than one identifier for the same item, use semicolons to separate them (e.g. in severe growth defects(A) Tetrad dissection of heterozygous diploid Y1F CTD strain (Y1F). Y1F CTD in haploid tetrads marked with NAT resistance and is present in all small-size tetrads. Yeast spot assay comparing growth of WT, Negatives, and two Y1F clones, with serial fivefold dilutions, on rich or synthetic total (SC) medium at 30C, or on rich Schisanhenol medium at indicated heat, for indicated quantity of days. (B) Cell-cycle assay showing unsynchronized cells stained with propidium iodide, the two peaks representing cells in G1/G0 and G2, respectively. (C) Western blot analysis of cell lysates derived from WT and Y1F strains using antibodies realizing the N-terminus of Rpb1 (y80), unphosphorylated consensus repeats (8WG16), Ser2P (3E10 and H5), Ser5P (3E8), Tyr1P (3D12) and GAPDH as indicated. (D) Western blot analysis of an Rpb1 co-IP (using y-80), probing for Ctk1 conversation (using 3HA tagged Ctk1 and HA antibody) with RNAP II (normalized to FLJ20032 Rpb3). 5% inputs shown. All results shown are representative of three impartial experiments. We next decided several properties of the Y1F strain, which we refer to just as Y1F. Comparing the growth of the parental strain (WT), consensus (CON), and Schisanhenol two individual Y1F (Y1F 2A and 2B) strains revealed that Y1F displayed a severe slow-growth defect on both rich and synthetic total media (Physique 1A). This phenotype was somewhat alleviated by growing at 37C, but, consistent with related results in fission yeast (Schwer et al. 2014), Y1F was chilly sensitive (Physique 1A). Tetrad analysis showed that this slow growth phenotype was specific to the Y1F mutation (Physique S1A), and the slow growth defect was also observed in liquid media (Physique S1B). Cell-cycle analysis showed that Y1F cells were defective in exiting G1/G0 and entering G2 (Physique 1B). We next examined accumulation of Rpb1 and CTD phosphorylation status by Western blot (Physique 1C). As expected, the WT strain showed evidence of Tyr1P as well Ser2P and Ser5P. However, Y1F Rpb1, while as expected was not detected by an anti-Tyr1P antibody (3D12), was also not recognized by antibodies specific for unphosphorylated consensus repeats (8WG16) or Ser2P (3E10). This likely indicates an failure of these antibodies to recognize the Y1F epitope (observe below). Indeed, a different Ser2P antibody (H5) produced a stronger transmission with the Y1F CTD, although whether this represents a bona fide increase in Ser2P, an increase in Ser2P with Ser5P (observe Phatnani and Greenleaf 2006), or differential acknowledgement due to an altered epitope, is not known. Ser5P levels appeared unaffected. Finally, analysis with an antibody (y-80) that recognizes an epitope in the first 80 residues of Rpb1 showed that Y1F Rpb1 accumulated to levels comparable to WT and CON Rpb1, and was Schisanhenol full length. This contrasts with the situation in vertebrate cells, where Y1F mutations resulted in CTD destabilization (Descostes et al. 2014; Hsin et al. 2014). Levels of the hyperphosphorylated Rpb1 IIo isoform were also comparable, suggesting that this apparent differences in Ser2P most likely reflected differences in epitope acknowledgement by the phospho-specific Abs. Supporting the conclusion that Ser2 phosphorylation was unaffected, a co-immunoprecipitation (co-IP) assay with WT or Y1F Rpb1 and Ctk1 revealed no changes in Ctk1 association with Rpb1, providing evidence that Ser2 phosphorylation by Ctk1 was not affected by the Y1F mutation (Physique 1D). Tyr1 interacts genetically with Mediator subunits MED13/CDK8/CYCC To investigate what pathways are affected by the Y1F mutation, we carried out a synthetic genetic array assay (SGA) (Tong et al. 2001, Rosonina et al. 2014). We performed the SGA as a suppressor assay, using a 5FOA-URA3 system with a plasmid-borne WT copy of Rpb1, comparing Y1F and CON strains (Poschke et al. 2012). Genes related to transcription found in each of two impartial suppressor SGA analyses are outlined in Physique 2A (the complete list is in Table S2), and those that best suppressed the Y1F slow growth phenotype are shown in Physique 2B. Sample tetrad dissections are shown in Physique S2A, and suppression of the Y1F growth defect could also be observed in liquid cultures (Physique S2B). Schisanhenol Interestingly, subunits of the Cdk8/CycC Mediator kinase module, including a protein required for stable association of Cdk8/CycC to Mediator (Med13), suppressed the Y1F phenotype when deleted, and were responsible for three of the four strongest interactions detected. The Cdk8/CycC kinase/cyclin pair inhibits transcription of numerous genes (many as part of the general stress response) and can phosphorylate transcription factors such as Gcn4 to dampen activation, in tandem with other post-translational modifications such as ubiquitination and SUMOylation (Bose et al. 2005; Rosonina et al. 2012; Allen and Taatjes 2015). The other strongest hit, Ubp8, is usually a component of the SAGA complex,.