A novel molecular switch.
Transcriptional regulation is a fundamental process for regulating the flux of all metabolic pathways. For the last several decades, the lac operon has served as a valuable model for studying transcription. More recently, the switch that controls the operon has also been successfully adapted to function in mammalian cells. Here we describe how, using directed evolution, we have created a novel switch that recognizes an asymmetric operator sequence. The new switch has a repressor with altered headpiece domains for operator recognition and a redesigned dimer interface to create a heterodimeric repressor. Quite unexpectedly, the heterodimeric switch functions better than the natural system. It can repress more tightly than the naturally occurring switch of the lac operon; it is less leaky and can be induced more efficiently. Ultimately, these novel repressors could be evolved to recognize eukaryotic promoters and used to regulate gene expression in mammalian systems.
Daber
Robert
R
Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, 19104-6059, USA.
Lewis
Mitchell
M
D000818:Animals; D019281:Dimerization; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins