Contacts between Tet repressor and tet operator revealed by new recognition specificities of single amino acid replacement mutants

Baumeister, Ralf; Helbl, Vera; Hillen, Wolfgang

doi:10.1016/0022-2836(92)91065-w

Cited by 45 publications

(54 citation statements)

References 51 publications

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“…3) and (288). In all TetR crystal structures elucidated to date (PDB identifiers: 2TCT; 2TRT; 1A6I; 1BJO; 1BJY; 1BJZ; 1ORK; and 1RP1), this repressor appears as a homodimer (29,30,159,183,(287)(288)(289)366). The TetR homodimer binds to the operator (Fig.…”

Section: Tetr Regulatormentioning

confidence: 99%

“…The following color code was used for complex networks: dark blue, TetR family member; orange, the gene directly regulated by the TetR family member; light blue, a regulator that modulates the expression of a TetR family member or which assists in the regulation of the gene under the control of a TetR family member; yellow boxes, signals and conditions influencing the system; open boxes, final results of the action of the system when the result is a scorable phenotype. References recommended for each circuit: panel A (29,38,286,288,388,417,418); panel B (4,7,13,31,35,110,176,191,230,245,270,436); panel C (8, 91, 201-204, 222, 290, 331); panel D (119,120,122,249,261,332,(350)(351); panel E (5,6,66,67,116,163); panel F (228,238,397,333,353,408); panel G (87-89, 125, 140, 214, 215, 295, 360, 403); panel H (48, 161); panel I (78); panel J (56,184,185,…”

Section: Beti Controls the Choline-glycine Betaine Pathway Of E Colimentioning

confidence: 99%

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The TetR Family of Transcriptional Repressors

Ramos

Martínez-Bueno

Molina‐Henares

et al. 2005

Microbiol Mol Biol Rev

988

1,133

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We have developed a general profile for the proteins of the TetR family of repressors. The stretch that best defines the profile of this family is made up of 47 amino acid residues that correspond to the helix-turn-helix DNA binding motif and adjacent regions in the three-dimensional structures of TetR, QacR, CprB, and EthR, four family members for which the function and three-dimensional structure are known. We have detected a set of 2,353 nonredundant proteins belonging to this family by screening genome and protein databases with the TetR profile. Proteins of the TetR family have been found in 115 genera of gram-positive, α-, β-, and γ-proteobacteria, cyanobacteria, and archaea. The set of genes they regulate is known for 85 out of the 2,353 members of the family. These proteins are involved in the transcriptional control of multidrug efflux pumps, pathways for the biosynthesis of antibiotics, response to osmotic stress and toxic chemicals, control of catabolic pathways, differentiation processes, and pathogenicity. The regulatory network in which the family member is involved can be simple, as in TetR (i.e., TetR bound to the target operator represses tetA transcription and is released in the presence of tetracycline), or more complex, involving a series of regulatory cascades in which either the expression of the TetR family member is modulated by another regulator or the TetR family member triggers a cell response to react to environmental insults. Based on what has been learned from the cocrystals of TetR and QacR with their target operators and from their three-dimensional structures in the absence and in the presence of ligands, and based on multialignment analyses of the conserved stretch of 47 amino acids in the 2,353 TetR family members, two groups of residues have been identified. One group includes highly conserved positions involved in the proper orientation of the helix-turn-helix motif and hence seems to play a structural role. The other set of less conserved residues are involved in establishing contacts with the phosphate backbone and target bases in the operator. Information related to the TetR family of regulators has been updated in a database that can be accessed at www.bactregulators.org

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Section: Tetr Regulatormentioning

confidence: 99%

Section: Beti Controls the Choline-glycine Betaine Pathway Of E Colimentioning

confidence: 99%

The TetR Family of Transcriptional Repressors

Ramos

Martínez-Bueno

Molina‐Henares

et al. 2005

Microbiol Mol Biol Rev

988

1,133

View full text Add to dashboard Cite

show abstract

“…Previous optimizations of the Tet system were based on the introduction of rationally designed mutations, and on directed evolution, in which large scale mutagenesis of the components of the Tet system was followed by functional screening of the mutants in bacterial or yeast assay systems (12)(13)(14)(15). However, these approaches are labor intensive, and mutations selected in a bacterial or yeast assay system may not be improvements in higher eukaryotes.…”

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confidence: 99%

Viral Evolution as a Tool to Improve the Tetracycline-regulated Gene Expression System

Das

Zhou

Vink

et al. 2004

Journal of Biological Chemistry

122

170

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We present viral evolution as a novel and powerful method to optimize non-viral proteins. We used this approach to optimize the tetracycline (Tc)-regulated gene expression system (Tet system) for its function in mammalian cells. The components of the Tet system were incorporated in the human immunodeficiency virus (HIV)-1 virus such that viral replication is controlled by this regulatory system. Upon long term replication of this HIV-rtTA virus in human T cells, we obtained a virus variant with an enhanced replication potential resulting from an improved rtTA component of the introduced Tet system. We identified a single amino acid exchange, F86Y, which enhances the transcriptional activity and doxycycline (dox) sensitivity of rtTA. We generated a new rtTA variant that is 5-fold more active at high dox levels than the initial rtTA, and 25-fold more sensitive to dox, whereas the background activity in the absence of dox is not increased. This new rtTA variant will be very useful in biological applications that require a more sensitive or active Tet system. Our results demonstrate that the viral evolution strategy can be used to improve the activity of genes by making them an integral and essential part of the virus.Technology for the regulation of gene expression in mammalian cells and tissues is of primary importance for a wide variety of basic and applied biological research areas, including functional genomics, gene therapy, animal models for human diseases, and biopharmaceutical protein production. All these applications require that production of the protein(s) of interest be regulated in both a quantitative and temporal way. For this purpose, artificial gene expression systems have been developed that are controlled by effector molecules in a dose-dependent and reversible manner. The most frequently used regulatory circuit is the so-called Tet system, which allows stringent control of gene expression by tetracycline (Tc) 1 or its derivative doxycycline (dox) (1-3). The Tet system is based on the specific, high affinity binding of the Escherichia coli Tet repressor protein (TetR) to the tet operator (tetO) sequence. Tc and dox induce a conformational change in TetR, which impedes the interaction with tetO. Fusion of the activation domain of the herpes simplex virus VP16 protein to TetR resulted in the transcriptional activator tTA, which induces gene expression from promoters placed downstream of tetO elements (P tet ) in eukaryotic cells. The presence of Tc or dox abolishes this gene expression. A tTA variant with four amino acid substitutions in the TetR moiety exhibits a reverse phenotype (4). This reverse tTA (rtTA) binds to P tet , and activates gene expression in the presence of dox but not in its absence. The Tet system is now widely applied to control gene expression in eukaryotes, including mammals, plants, and insects (reviewed in Ref. 1). Since the Tet system originates from a bacterial regulatory system, it seems likely that the components can be optimized for their new transcriptional function i...

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“…Thorough structure-function analyses of the interaction between TetR and tetO have revealed (19,20) that some mutations introduced into the helix-turn-helix motif of TetR that abolish the binding to the tetO can be compensated by alterations within the operator sequence. Two mutant repressor͞operator combinations exhibited particularly remarkable properties.…”

Section: Resultsmentioning

confidence: 99%

Generation of conditional mutants in higher eukaryotes by switching between the expression of two genes

Baron

Schnappinger

Helbl

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

112

108

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A regulatory system for the in-depth study of gene functions in higher eukaryotic cells has been developed. It is based on the tetracycline-controlled transactivators and reverse tTA, which were remodeled to discriminate efficiently between two different promoters. The system permits one to control reversibly the activity of two genes, or two alleles of a gene, in a mutually exclusive way, and also allows one to abrogate the activities of both. This dual regulatory circuit, which can be operated by a single effector substance such as doxycycline, overcomes limitations of conventional genetic approaches. The conditional mutants that can now be generated will be useful for the study of gene function in vitro and in vivo. In addition, the system may be of value for a variety of practical applications, including gene therapy.

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Contacts between Tet repressor and tet operator revealed by new recognition specificities of single amino acid replacement mutants

Cited by 45 publications

References 51 publications

The TetR Family of Transcriptional Repressors

The TetR Family of Transcriptional Repressors

Viral Evolution as a Tool to Improve the Tetracycline-regulated Gene Expression System

Generation of conditional mutants in higher eukaryotes by switching between the expression of two genes

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