Figure 5.—

Interaction of various Dpb2p mutants with Pol2p C terminus by two-hybrid assay. (A) Two-hybrid interactions. The Y190 strain was transformed with indicated plasmids and the ability of particular Dpb2p variants to interact with the C terminus of Pol2p was tested using the lacZ genetic reporter. Strains carrying empty or bacterial polymerase dnaE gene-coding plasmids were used as negative controls, whereas the strain bearing the wild-type GAL4 gene served as a strong positive control. The β-galactosidase expression levels were determined as described in materials and methods. Plate assays were carried out as described by Vojtek et al. (1993), while the in vitro assay was conducted according to Rose et al. (1990). (B) Expression of BD_Gal4p-Dpb2p fusion variants. The Y190 strain bearing the indicated plasmids for the two-hybrid assay was prepared and Western blotting was performed as described in materials and methods. The indicated Dpb2p variants are BD_Gal4p fusions and Pol2p_{(K2090-I2222)} is an AD_Gal4p fusion. (Top) Blots probed with anti-BD_Gal4p antibodies. The 97-kDa band indicates the BD_Gal4p-Dpb2p fusions. (Bottom) The same blots probed with anti-Hts1p (histidyl-tRNA synthetase, 60 kDa; Chiu et al. 1992) antibodies as a gel loading control.