FIG. 4.

Sub1 influences recruitment and kinase activity of the elongation kinase Ctk1 during transcription. (A) sub1Δ and ctk1Δ are synthetically lethal. A diploid yeast strain heterozygous for both SUB1 and CTK1 (sub1Δ::URA3/SUB1 ctk1Δ::kanMX/CTK1) was sporulated, and the meiotic progeny were separated by tetrad dissection and allowed to grow for 3 days. Thirteen tetrads were dissected, with nine showing a tetratype segregation pattern (eight of which are shown), and two showing a paternal ditype segregation pattern. The genotype of the resulting colonies was inferred by growth or lack of growth on selective medium. Cells with a deletion of CTK1 alone show a slow-growth phenotype, as reported previously. (B) SUB1 deletion causes increased Ctk1 kinase activity. Whole-cell extracts were prepared from wt and sub1Δ strains with an HA-tagged Ctk1, and in vitro kinase assay was performed as described in the Fig. 1 legend to analyze CTD Ser2 phosphorylation, using anti-CTD Ser2P. (C) Schematic representation of the ACT1, PYK1, and PMA1 genes. Numbers are nucleotide positions relative to start codon (+1), and black bars represent PCR products analyzed by ChIP. (D) Increased Ctk1-HA association with chromatin in cells lacking SUB1. ChIP for Ctk1-HA was performed in wt and sub1Δ cells. Ctk1-HA association with PMA1, PYK1, and ACT1 genes was analyzed by qRT-PCR, and quantifications were graphed (see Materials and Methods). (E) Ctk1-HA/Rpb1 ratio. Ctk1-HA and Rpb1 cross-linking were independently quantified in wt and sub1Δ cells, and then Ctk1/Rpb1 ratio was calculated and graphed. Error bars show standard deviations.