Investigation of changes in tetracycline repressor binding upon mutations in the tetracycline operator

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DOIResolve DOI: http://doi.org/10.1021/je500225x
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TypeArticle
Journal titleJournal of Chemical and Engineering Data
ISSN0021-9568
Volume59
Issue10
Pages31673176; # of pages: 10
AbstractThe tetracycline operon is an important gene network component, commonly used in synthetic biology applications because of its switch-like character. At the heart of this system is the highly specific interaction of the tet repressor protein (TetR) with its cognate DNA sequence (tetO). TetR binding on tetO practically stops expression of genes downstream of tetO by excluding RNA polymerase from binding the promoter and initiating transcription. Mutating the tetO sequence alters the strength of TetR-tetO binding and thus provides a tool to synthetic biologists to manipulate gene expression levels. We employ molecular dynamics (MD) simulations coupled with the free energy perturbation method to investigate the binding affinity of TetR to different tetO mutants. We also carry out in vivo tests in Escherichia coli for a series of promoters based on these mutants. We obtain reasonable agreement between experimental green fluorescent protein (GFP) repression levels and binding free energy differences computed from molecular simulations. In all cases, the wild-type tetO sequence yields the strongest TetR binding, which is observed both experimentally, in terms of GFP levels, and in simulation, in terms of free energy changes. Two of the four tetO mutants we tested yield relatively strong binding, whereas the other two mutants tend to be significantly weaker. The clustering and relative ranking of this subset of tetO mutants is generally consistent between our own experimental data, previous experiments with different systems and the free energy changes computed from our simulations. Overall, this work offers insights into an important synthetic biological system and demonstrates the potential, as well as limitations of molecular simulations to quantitatively explain biologically relevant behavior.
Publication date
PublisherACS Publications
LanguageEnglish
AffiliationNational Research Council Canada; Human Health Therapeutics
Peer reviewedYes
NPARC number21275472
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Record identifier71c9cab7-718e-43b3-b6e8-35c32f2aaa35
Record created2015-07-14
Record modified2016-05-09
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