AraC is composed of 292 amino acid residues and uses the active sites formed by the amino acids 198-219 and 246-269 to bind to DNA. The isolated AraC protein binds to araO1(-100 to -144), acting as an inhibitor. When the C protein binds with the inducer AraBAD, forming a complex, it binds to the araI region (-40 to -78) allowing RNA Pol to bind to the PBAD site (+140) and transcribe downstream genes.

TetR monomer is made up of 198 amino acid residues and, when dimerized, is folded by 10 alpha-helices connected by turns and loops. The three-dimensional structure of the TetR monomer is mainly stabilized by hydrophobic inverse helices. The sequence of amino acids 33-52 located at the N-terminal is the DNA binding region, composed of helices a1, a2, a3, and their symmetric helices a1', a2', a3'. The regulatory core, composed of helices a5, a10 and their symmetric helices a5', a10', controls dimerization and the binding sites of each monomer in the presence of divalent cations. Helices a5, a8, a10, and their corresponding helices form the core scaffold, and their structure is the most conserved in the entire TetR conformation.

To avoid significant protein structural changes, we only subjected the DNA binding sites of AraC and TetR to computer-simulated random mutations. The optimized proteins obtained showed a slight change in their DNA binding site structures through structural simulations, while the rest of the structures remained largely unchanged. (Figure 6, Figure 7).

Comparison of AraC homologous modeling results. (a) Pre-mutation AraC structure (b) Homologous modeling structure of post-mutation AraC. AraC rated P from 12.98 to 15.94 . The blue part is the DNA-binding site of the protein, the yellow part is the rest of the structure, and the red part represents the amino acid after the mutation.

Comparison of TerR homologous modeling results. (a) Pre-mutation TetR structure (b) Post-mutation TetR homologous modeling structure. The score P of TetR ranges from 5.38 to 8.19. The blue part is the DNA-binding site of the protein, the yellow part is the rest of the structure, and the red part represents the amino acid after the mutation.