Fig 5.

Phosphorylation by CK2 negatively regulates Spt2 chromatin function. (A and B) Mutations of CK2 sites to nonphosphorylatable residues do not result in an Spt⁻ phenotype. Patches show the growth of an spt2Δ mutant containing the lys2-128δ Spt reporter and transformed with a plasmid carrying either the wild type or a mutated version of SPT2, as indicated. The photographs were taken after 1 or 2 days of incubation at 30°C. (C) Mimicking phosphorylation of Spt2 at CK2 RII or RI/RII leads to an Spt⁻ phenotype. Serial dilutions of the spt2Δ mutant culture containing the lys2-128δ Spt reporter and transformed with a plasmid carrying either wild-type SPT2 or the indicated mutated version of SPT2 were grown on synthetic complete medium lacking uracil (SC−URA) or on synthetic complete medium lacking both uracil and lysine (SC−URA−LYS). The photographs were taken after 2 days of incubation at 30°C. (D) An spt2 mutant mimicking RI/II CK2 phosphorylation is synthetically lethal with the paf1Δ mutant. An spt2Δ mutant containing a CEN URA3 SPT2 vector was transformed with a second CEN LEU2 plasmid containing wild-type SPT2 or the indicated mutated version of SPT2. This strain was then mated with a paf1Δ mutant, diploids were sporulated and dissected, and representative progeny containing both plasmids were spotted onto medium lacking uracil or medium containing 5-fluoroorotic acid (5 FOA). The photograph was taken after 2 days of incubation at 30°C. (E) An spt2 mutant mimicking RI/II CK2 permanent phosphorylation has synthetic interactions with a cdc73Δ mutant. An spt2Δ cdc73Δ mutant transformed with a CEN URA3 plasmid containing wild-type SPT2 or the indicated mutated version of SPT2 was spotted onto medium lacking uracil at 30°C or 37°C for 2 days.