Allostery and applications of the lac repressor

摘要: The lac repressor has been extensively studied for nearly half a century; this long and complicated experimental history leaves many subtle connections unexplored. This thesis sought to forge those from isolated and purified components up to functioning genetic switches in cells even organisms. We first connected the genetics structure of the vivo gene regulation Escherichia coli. We found that point mutations amino acids structurally make specific contacts with DNA can alter repressor-operator affinity conformational equilibrium repressor. then that structurally contacts with effector molecules repressor-effector affinity conformational equilibrium. All results are well explained by Monod, Wyman, Changeux model allostery. next vitro components E. coli. used an vitro transcription assay measure binding affinity, repressor-effector binding affinity, Only affinity disagreed literature values other experiments, however it did agree with a published value which should hold under conditions. were able use our in thermodynamic parameters accurately predict when cell crowding was considered. Finally we developed autogenously regulated lac repressor AAV-mediated therapy. improve regulation of autogenous switch using multiple operator sites, tetrameric lac repressor, shown to function various cell types capable reversible luciferase in living mice.