We report for the first time the in vitro characterization of a reverse tetracycline repressor (revTetR). The dimeric wild-type repressor (TetR) binds to tet operator tetO in the absence of the inducer anhydrotetracycline (atc) to confer tight repression. We have isolated the revTetR G96E L205S mutant, which, contrary to TetR, binds tetO only in the presence of atc. This reverse acting mutant was overproduced and purified. Effector and DNA binding properties were analyzed by EMSA and quantified by fluorescence titration and surface plasmon resonance. The association constant K(A) of revTetR for binding of [atcMg](+) is approximately 10(8) M(-1), four orders of magnitude lower than that of TetR. The affinity of TetR for tetO is 5.6 +/- 2 x 10(9) M(-1) and that for revTetR in the presence of atc is 1 +/- 0.2 x 10(8) M(-1). Both induced forms, the atc-bound TetR and the free revTetR, have the same low affinity of 4 +/- 1 x 10(5) M(-1) for DNA. Therefore, atc does not act as a dimerization agent for revTetR. We discuss the structural differences between TetR and revTetR potentially underlying this reversal of activity.
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http://dx.doi.org/10.1093/nar/gkh200 | DOI Listing |
J Inorg Biochem
January 2025
Department of Chemistry, University of South Florida, Tampa, FL 33620-5250, USA; Institute of Drug Discovery Technology, Ningbo University, Ningbo, Zhejiang 315211, PR China. Electronic address:
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Department of Psychiatry and Behavioral Science, Tokyo Metropolitan Institute of Medical Science, Tokyo, 156-8506, Japan.
J Cell Sci
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National Institute of Biological Sciences, Beijing 102206, China.
The ability to manipulate gene expression is valuable for elucidating gene function. In the fission yeast Schizosaccharomyces pombe, the most widely used regulatable expression system is the nmt1 promoter and its two attenuated variants. However, these promoters have limitations, including a long lag, incompatibility with rich media and unsuitability for non-dividing cells.
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University of Newcastle, Newcastle, Australia.
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Key Laboratory of Major Diseases in Children, Ministry of Education, National Clinical Research Center for Respiratory Diseases, National Key Discipline of Pediatrics, Laboratory of Infection and Microbiology, Beijing Pediatric Research Institute, Beijing Children's Hospital, Capital Medical University, National Center for Children's Health, Beijing 100045, China. Electronic address:
Antimicrobial resistance has been an increasingly serious threat to global public health. The contribution of non-antibiotic pharmaceuticals to the development of antibiotic resistance has been overlooked. Our study found that the anti-inflammatory drug phenylbutazone could protect P.
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