Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 391, Issue 4, Pages 661-670Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2009.06.039
Keywords
directed evolution; repression; heterodimer; chimeric operator
Categories
Funding
- NIGMS NIH HHS [T32 GM008275, R01 GM044617-17, R01 GM044617] Funding Source: Medline
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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. (C) 2009 Elsevier Ltd. All rights reserved.
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