FIG 2.

ThiR structure and identification of its target promoters. (A) ThiR domain architecture and 3D structural models. Data under PDB accession numbers 1LMB and 2PB9 and other homologs were used as the template to build the model. The ribbon diagrams include the N-terminal HTH domain of ThiR (dark red) overlaid with the lambda repressor-operator complex (brown) (PDB accession number 1LMB), represented as a dimer (left), and the ThiN domain of ThiR (light blue) overlaid with the ThiN domain of Methanocaldococcus jannaschii ThiDN (brown) (PDB accession number 2PHP) (right). (B) A reporter gene (bgaH) assay was performed by using wild-type (wt) and ΔthiN (ΔthiR) mutant cells carrying transcriptional fusion constructs of promoters. Cells were grown to log phase in GMM with and without thiamine, THZ, or HMP, as indicated. The cell lysate (5 μg of total protein) was used in the assay. Experiments were performed in triplicate (n = 3), and data represent mean results ± standard errors of the means. P values were determined by two-tailed, unpaired Student's t test (*, P < 0.05; **, P < 0.01). See Materials and Methods for details.