FIG. 5.

Mutations in RFXANK and RFXAP genes in BLS prevent the assembly of the RFX complex on DNA. (A) Analyses of interactions between the three RFX proteins and DNA. Wild-type RFXANK, RFXAP, and RFX5 proteins, mutant RFXAP(1-243) protein, and mutRFXANK were transcribed and translated in vitro using the rabbit reticulocyte lysate system. Different combinations of proteins were incubated with the ³²P-labeled oligonucleotide containing S and X boxes from the DRA promoter (SX oligonucleotide). Two different unlabeled competitor oligonucleotides were used, the specific SX oligonucleotide and the nonspecific SRE oligonucleotide (lanes 8 and 9). The rabbit reticulocyte lysate alone did not bind to the SX oligonucleotide (data not shown). (B) Amounts of proteins used in EMSA. Presented are inputs of the ³⁵S-labeled proteins that were transcribed and translated in vitro using the rabbit reticulocyte lysate in parallel with their unlabeled counterparts: RFXAP (lane 1), RFXANK (lane 2), RFXAP(1-243) (lane 3), RFX5 (lane 4), and mutRFXANK (lane 5).