Probing the Informational and Regulatory Plasticity of a Transcription Factor DNA–Binding Domain
(A) A schematic representation of the MarA selection plasmid. Expression of the marA gene is controlled by an AraC repressed, L-arabinose inducible promoter . Unique restriction sites (light orange arrows) flank the promoter region of the tetracycline resistance gene tet and helix 3 of the marA gene. Promoter and binding domain variants can be cloned into this plasmid and functional binding domain-binding site pairs can be identified by selection in tetracycline+L-arabinose. (B) The sequence of the MarA-activated tet promoter (top) and a cartoon marking each component (bottom). This construct is based on the promoter used in . Bases that were varied in binding domain selection experiments are designated by a blue ‘N’. Additional bases that were randomized in the binding site selection are shown in purple. Orange boxes mark the restriction sites used to clone in these constructs. The mar binding site has the opposite orientation as in Figure 1. (C) The sequence of the MarA binding domain variants (top) and a cartoon marking components within this region. The three residues that were randomized are marked with yellow boxes. The boundaries of helix 3 are marked with red boxes.