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

FIG. 5.

FIG. 5.

Bur1 kinase recruitment and activity is also enhanced by deletion of SUB1. (A) SUB1 deletion increases the elongation defect of bur1 mutants. Phenotypic analysis of SUB1-BUR1 genetic interaction. SUB1 was deleted in a bur1-2 strain and in the isogenic wt strain. In the case of the bur1-23 mutant, we generated a diploid strain by crossing it with a sub1Δ mutant. The diploid was sporulated, and the tetrads dissected and analyzed. The growth phenotype of one tetrad is shown. Yeast strains with the indicated genotypes were spotted on yeast extract-peptone-dextrose or synthetic complete (SC) medium containing 50 μg/ml of 6-azauracil (6-AU), and plates were incubated for 3 days. As shown, SUB1 deletion increases the growth and elongation defects of bur1-23 cells and the elongation defect of bur1-2 cells. (B) sub1Δ cells are sensitive to 6-AU in liquid medium. Growth curves of wt and sub1Δ strains in SC medium without or with 75 μg/ml of 6-AU. (C) Bur1 kinase activity is increased in sub1Δ mutant. In vitro kinase assay was performed as described in the Fig. 1 legend in wt and sub1Δ cells expressing an HA-tagged Bur1. CTD phosphorylation was analyzed using anti-CTD Ser5P (CTD4H8, top left), anti-Ser2P (ab5095, top right) and anti-nonphosphorylated CTD (8WG16) antibodies. Bur1-HA levels were tested with anti-HA antibody. For both Ser2P and Ser5P, we observe increased phosphorylation of the CTD in the absence of Sub1. (D) SUB1 deletion increases Bur1 autophosphorylation and Bur1/RNAP II-CTD interaction. Whole-cell extracts were prepared from wt and sub1Δ Bur1-HA strains. Epitope-tagged kinase complexes were immunoprecipitated with 12CA5-protein A beads, and kinase activity with or without ATP was assayed. SDS-PAGE and immunoblot analysis were performed to analyze Bur1 autophosphorylation using HA antibody and CTD Ser5P/Bur1-HA coimmunoprecipitation using anti-Ser5P (CTD4H8) antibody. (E) SUB1 deletion slightly influences Bur1 association with coding gene regions compared to its influence on Rpb1 association. Bur1 occupancy at PMA1, PYK1, and ACT1 genes was assayed by ChIP in wt and sub1Δ cells. Graph shows qRT-PCR quantifications performed as described in the Fig. 4 legend.