Skip to main content
. 2019 May 2;39(5):BSR20182383. doi: 10.1042/BSR20182383

Figure 5. Mutations of TFIIAγ gene repressed TFIIAγ binding to the TATA-containing promoters.

Figure 5

(A) Coomasie staining showing the effect of expression and purification for human recombinant TBP. Recombinant TBP was expressed in E. Coli BL21 (DE3), purified using nickel agarose and detected by SDS–PAGE and Coomasie staining. (B) Coomasie staining showing the effect of purification for recombinant TFIIAγ and its mutants. Recombinant WT TFIIAγ and its mutants were prepared and detected as described in (A). (C, D) Mutations of TFIIAγ gene reduced TFIIAγ binding to the AdML promoter in vitro. Immobilized DNA-protein binding assays were performed using the biotin-modified AdML core promoter and recombinant proteins TBP and WT TFIIAγ or its mutants. The DNA-binding protein was detected by Western blot and the antibodies against TBP or TFIIAγ (C). Density of the bands in (C) was quantified and shown in (D). (E) ChIP-qPCR showing the effect of the N63A mutant on the occupancies of components of the Pol II transcription machinery at the IGF2 promoter. (F) ChIP-qPCR showing the effect of the N63A mutant on the occupancies of components of the Pol II transcription machinery at the IGF2 promoter. Each column in (E) and (F) represent the mean ± S.E.M. of three independent experiments.*, P<0.05; **, P<0.01; P-values were obtained with one way ANOVA.