Figure 5.

EMSA to detect the association of TFIIA with TATA the box. (A) in vitro processing of recombinant TFIIAαβ precursor protein. Twenty nanograms of purified recombinant TFIIAαβ and DGAA expressed in E. coli were mixed with 70 ng of purified recombinant FH-Taspase1 and incubated at 37°C for 1 h. Proteins were separated by SDS-PAGE and detected by silver staining. (B–D) EMSA of TFIIA and TBP to detect TATA box binding of the GAPDH promoter. Purified TFIIAαβ, His-TFIIAγ and FH-TBP were used. Panel B: (a) processed TFIIAαβ (indicated as Processing +, same with αβ of panel b) and unprocessed TFIIAαβ (indicated as Processing -, same with α/β of panel b) were used for EMSA. Processing: purified recombinant TFIIAαβ and DGAA were incubated with FH-Taspase1, and TFIIA proteins were purified. (b) Indicated combinations of purified proteins were used for EMSA. Panel C: cold probe DNA (WT) and its mutant (mut) were used as competitors in EMSA binding reactions. Sequences of wild-type and mutant competitors were 5′-CGGTTTCTATAAATTGAGCC and 5′-CGGTTTCCAGTAACTGAGCC, respectively. Panel D: specific antibodies against TBP, TFIIAαβ and control IgG were included in the EMSA. αβ and α/β indicate unprocessed and processed TFIIAαβ, respectively.