Figure 6.

Aft1p and Aft2p bind to the same promoter fragment in vitro but differentially regulate gene expression. (A) S1 nuclease protection assays to quantify mRNA levels of genes activated by the AFT1-1^up and AFT2-1^up alleles. RNA was isolated from the aft1Δaft2Δ strain transformed with a control plasmid (−) (pRS316 in the case of pAFT1-1^up and pRS416 in the case of pAFT2-1^up), pAFT1-1^up (1^up), and pAFT2-1^up (2^up) and grown in CMD(−Ura) medium to mid-log phase. The Upper band for each sample is the specified gene, and the Lower band the calmodulin loading control (CMD1). (B) Gel retardation assays were performed by using a ³²P-labeled 30-bp duplex of the FET3 promoter as probe with no protein (lane 1), and lysates from E. coli containing pET3 (lane 2), pAft1-313 (lane 3), and pAft2-214 (lane 4).