Fig. 4.
Dissociation of Maf1 from tRNA genes following transfer of yeast from nonfermentable glycerol growth conditions to glucose is dependent on CK2 activity. Relative occupancy of Maf1 at selected class III genes. Strain expressing Myc-tagged Maf1 was grown in rich glucose medium (YPD) to exponential phase and transferred to a nonfermentable glycerol medium (YPGly). Following 3-h incubation at 30 °C, the culture was split in half; one part was transferred to YPD with 2% (vol/vol) DMSO and the second, to YPD with 200 μM TBBt in DMSO. (A) Cross-linked chromatin was immunoprecipitated with antibodies against Myc epitope, followed by quantitative real-time PCR. Occupancies of Maf1 at tRNA −Leu, tRNA −Trp, and 5S rRNA genes were measured relative to occupancy at 35S rRNA. (B) Cellular extracts were incubated with magnetic beads coated with anti-HA monoclonal antibody followed by elution of bound proteins and Western analysis with anti-HA and anti-Maf1 antibodies. (C) Quantification of the amounts of Maf1 immunopurified with Rpc160. Experiment was performed in triplicate to estimate SD.