Figure 3

Expression and binding of DNA-binding domains (DBD) of putative repressor proteins to the AGAA-box within xyn2 promoter of H. Jecorina. (A) SDS-PAGE of two clones of the DBD of 3151, three clones of the DBD of 7236, and two clones of the DBD of 2488. All clones heterologously expressed the GST-fusion proteins (3151prp, 7236prp, 2488prp). GST, expression of the GST-protein alone, as a control. A prestained protein ladder (Fermentas) was used. (B) EMSA with 100 ng of the DBDs expressed as GST-fusion proteins (see Fig. 3A). Finally, 15 ng of labelled oligonucleotides, one covering the respective area of the xyn2 promoter (Lpxyn2, see Table 1) and the other bearing a mutation in the AGAA-box (from AGAA to CTCC, Lpxyn2 M, see Table 1), were assayed alone (lanes 1, 2), with GST alone (negative control, GST), or with the thrombin-cleaved DBDs (3151prp_DBD, 7236prp_DBD, 2488prp_DBD).