Figure 2. The Core Domain of p53 Mediates the p53-Pol II Interaction in Whole Cell Extracts.

(A) Domain structures of p53⁺ and p53^coreΔ. TA1 and TA2, transactivation domains 1 and 2; Tetra., tetramerization domain; Reg., regulatory domain. (B) p53 immunoblot analysis of cells expressing p53⁺ (strain SY1000) and p53^coreΔ (strain SY1004) regulated by the GAL1 promoter. Cells were grown in rich medium containing 1.8% raffinose and 0.2% galactose. p53 was detected through use of the DO-1 mAb; expression levels were internally normalized to those of Pgk1. (C) The Pol II large subunit co-immunoprecipitates with p53⁺, but not with p53^coreΔ, in yeast WCEs. Immunoprecipitates were obtained from WCEs isolated from strains SY1000 and SY1004 cultivated as above using Myc- or p53-specific antibodies (9E10 or DO-1, respectively) (lanes 1–4). Rpb1 was detected using anti-CTD antiserum. Lanes 1 and 3 serve as negative controls as cells do not express a Myc-tagged protein. (D) p53⁺, but not p53^coreΔ, co-IPs with Pol II. Immunoprecipitates were generated using either pre-immune or anti-Pol II (CTD) antiserum (lane 1–4) from extracts as above. p53 and p53^coreΔ were detected using DO-1.