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. 2010 Sep 7;30(21):5180–5193. doi: 10.1128/MCB.00819-10

FIG. 3.

FIG. 3.

SUB1 genetically interacts with SRB10, and SUB1 deletion negatively influences Srb10 kinase activity and association with active genes. (A) Genetic interaction between SUB1 and SRB10. SUB1 deletion partially suppresses the slow-growth phenotype of srb10Δ cells and overexpression of SUB1 enhances it, as shown by spot assay. To overexpress SUB1, its open reading frame was cloned under the control of the ADH1 promoter in a plasmid that was transformed into the srb10Δ strain (ADH1SUB1). Yeast strains with the indicated genotypes were spotted onto synthetic complete medium and grown at 30 and 37°C for 2 days. (B) Results of in vitro CTD kinase assay showing that SUB1 deletion causes a decrease in CTD phosphorylation by Srb10. Whole-cell extracts were prepared from the wt and sub1Δ strains expressing MYC-tagged Srb10, and in vitro kinase assay and CTD phosphorylation analysis were performed as described in the Fig. 1 legend. CTD Ser5 and Ser2 phosphorylation was analyzed using the following antibodies: CTD4H8 (anti-CTD Ser5P), ab5095 (anti-CTD Ser2P), 8WG16 (anti-CTD), anti-MYC antibody (Srb10 levels), and anti-PGK antibody (PGK levels, as control). (C) Induction of GAL1 transcription was monitored by RT-PCR. Total RNA was isolated from wt and sub1Δ cells grown under noninducible (2% raffinose [RAF]) and inducible (2% galactose [GAL]) conditions. cDNA was synthesized, and PCR performed using specific primers for GAL1, PYK1, SUB1, and SRB10 genes. (D) ChIP analysis of Srb10 was conducted with wt and sub1Δ cells grown in raffinose- or galactose-containing medium, using anti-MYC antibody. Srb10-MYC cross-linking to the promoter of PYK1 and to the upstream activating sequence of the GAL1 (UAS-GAL1) gene was analyzed by qRT-PCR. Quantifications of the results are shown in the graph, where numbers on the y axis represent the ratio of the values obtained from specific primer products to the value for the negative control (intergenic region of chromosome VII), after normalizing to the results for the input controls. Error bars show standard deviations. (E) SRB10 deletion causes an increase in CTD Ser5 phosphorylation by Kin28, reflecting increased Kin28 levels in the srb10Δ cells. Whole-cell extracts were prepared from wt and srb10Δ strains expressing HA-tagged Kin28, and in vitro kinase assays and CTD phosphorylation analysis were performed. CTD phosphorylation was analyzed using the following antibodies: CTD4H8 (anti-CTD Ser5P) and 8WG16 (anti-CTD). Kin28-HA and PGK levels were analyzed using 12CA5 (anti-HA) and anti-PGK, respectively.