FIG 6.

Binding of RamB to the icl1 regulatory region. (A) DNase I footprinting analysis of the icl1 regulatory region bound by purified RamB. The DNA fragments containing the coding and noncoding strands labeled with TAMRA at their 5′ ends were incubated with increasing concentrations of purified RamB (0.3, 0.6, and 0.9 μM) in the absence (- Suc-CoA) or presence (+ Suc-CoA) of 200 μM succinyl-CoA and then subjected to DNase I footprinting reactions. The amounts of RamB protein used are given above the lanes. The regions protected by RamB, which indicate the RamB-binding sites, are marked by thick black lines (RamBS1, RamBS2, and RamBS3). Lanes G, A, T, and C represent the sequence ladders. (B) The upstream sequence of the icl1 gene, including its putative promoter region and the identified cis-acting regulatory elements involved in the regulation of the icl1 gene. The RamB-binding sites (RamBS1, RamBS2, and RamBS3) are indicated in green font. The nucleotides in RamBS1 and RamBS2 which were mutated for the promoter assay in Fig. 7 are indicated with asterisks. The TSP of the icl1 gene is marked by +1. The putative promoter region (−35 and −10) of the icl1 gene is boxed. The start codon of icl1 is indicated both in red font and by the arrow indicating the transcriptional direction. The numbers on the left side of the sequences show the positions of the leftmost nucleotides relative to the TPS of the icl1 gene.