Skip to main content
. 2004 Jul 15;23(15):3083–3091. doi: 10.1038/sj.emboj.7600304

Figure 2.

Figure 2

Mutational analysis of the TFIIAαβ cleavage region. (A) Sequence alignment indicates conservation of the GTG/DT TFIIAαβ cleavage site (marked by arrow) among human, mouse, Xenopus, Drosophila and S. pombe. The 12 residues following the arrow were from cycles 1 to 12 in the Edman degradation. The amino acids of the CRS that are essential for cleavage are boxed. (B) Extracts from U2-OS cells transfected with plasmids expressing Myc-tagged hTFIIAαβ (wt or the Ala mutants of the indicated residues) and hTFIIAγ were analysed by SDS–PAGE and immunoblotting using antibodies against TFIIAα (upper panel) and TFIIAβ and TFIIAγ (lower panel). The asterisk marks the position of residue A267 that was not included in the analysis. (C) U2-OS cell extracts, from panel B were subjected to immunoprecipitation using an antibody against TFIIAγ. The immunoprecipitates were analysed by SDS–PAGE and immunoblotting as in (B). (D) Whole-cell extracts (WCE) from U2-OS cells transfected with plasmids expressing hTBP, Myc-tagged hTFIIAαβ (wt or the indicated mutants) and hTFIIAγ as indicated were used for EMSA with a synthetic oligonucleotide comprising the adenovirus 2 major late TATA box (lanes 4–13). Recombinant TBP and TFIIA were used giving rise to the DA (TBP–TFIIA–DNA) complex (lane 3).