The TetR family of transcriptional repressors

Abstract

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, alpha-, beta-, and gamma-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.

FIG. 1.

Alignment of 42 members of the TetR family that exemplify the TetR family profile. The blue column indicates α-helix residues involved in DNA contacts in the crystal structure of TetR and QacR. The yellow column indicates turns. The most conserved residues are shaded. Abbreviations are as follows: BACME, Bacillus megaterium; BACSU, Bacillus subtilis; ECOLI, Escherichia coli; HAEIN, Haemophilus influenzae; LACLA, Lactobacillus lactis; MYCTU, Mycobacterium tuberculosis; RHIME, Rhizobium meliloti; STRGA, Streptomyces sp.; VIBHA, Vibrio haemophilus; XANAU, Xanthomonas sp.

FIG. 2.

Ribbon diagram of a TetR homodimer. Monomers are shown in blue or red. Two tetracycline molecules, each bound to a monomer, are shown in grey. α-Helices 2 and 3 in the blue monomer and α2′ and α3′ in the red monomer constitute the shared HTH DNA binding domain. α-Helix 1 and part of helix α-4, together with α-helices 2 and 3, comprise the sequence that best defines the TetR family profile. (Adapted from Hinrichs et al. [150] with permission of the publisher.)

FIG. 3.

Binding of TetR to its operator site. A) tetR operator and contact regions. The tetR operator is a palindromic sequence. Horizontal bars show nucleotides contacted by each monomer of the TetR dimer. B) Interaction of TetR residues with specific nucleotides (arrows) and phosphate backbone (blue lines) in the operator region. The amino acids involved in DNA binding extend from residues 27 to 48. Contacts established with the operator were confirmed by footprint assays, by analysis of TetR mutants, and by crystallographic studies (29, 30, 159, 266, 366). C) Representation of each homodimer bound to the tet operator in a double-helix representation. (Adapted from Orth et al. [288] with permission of the publisher.)

FIG. 4.

Representation of the TetR cavity involved in the binding of tetracycline. Left) In the absence of tetracycline. Right) In the presence of tetracycline. The green ball represents the Mg²⁺ ion. Specific interactions are not drawn for the sake of clarity but are described in the text. (Adapted from Orth et al. [287, 289] and Kisker et al. [183] with permission of the publishers.)

FIG. 5.

Docking of a TetR monomer without (grey) and with (red) tetracycline. Note the alterations induced in the α₁-α₃ region involved in binding to the target operators. The increase in distance between α3 and α3′ with tetracycline results in the inability of TetR to maintain the specific interactions shown in Fig. 3, and therefore the repressor is released. (Adapted from Orth et al. [288] with permission of the publisher.)

FIG. 6.

Binding of QacR to its operator site. A) Interaction of QacR with the qac operator. B) Contacts established by residues at α-helix 3 of QacR homodimers A and B with specific nucleotides (arrows) and phosphate backbone (blue lines) in the synthetic operator used for QacR-DNA cocrystal (349, 350). C) Representation of the two QacR homodimers bound to the qac operator in a double-helix representation. (Adapted from Schumacher et al. [351] with permission of the publisher.)

FIG. 7.

Ribbon representation of the two QacR homodimers bound to target DNA in a double-helix representation (A) and details of the contacts established by α-helix 3 of monomers of different homodimers when recognizing overlapping sites (B). (Adapted from Schumacher et al. [351] with permission of the publisher.)

FIG. 8.

Regulatory networks involving members of the TetR family. Although TetR and QacR cannot be considered part of a network, their type of control is shown because it is frequently found in members of the family. 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, 208, 413); panel K (133, 134, 158, 413); panel L (127); panel M (61, 266); panel N (240, 241, 345); panel O (248); panel P (59, 63, 242, 243, 355); panel Q (23, 24, 51, 107, 232, 255); panel R (97, 107, 117, 252, 341, 363); and panel S (107, 117, 181, 196, 217, 247).

FIG. 8.

Regulatory networks involving members of the TetR family. Although TetR and QacR cannot be considered part of a network, their type of control is shown because it is frequently found in members of the family. 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, 208, 413); panel K (133, 134, 158, 413); panel L (127); panel M (61, 266); panel N (240, 241, 345); panel O (248); panel P (59, 63, 242, 243, 355); panel Q (23, 24, 51, 107, 232, 255); panel R (97, 107, 117, 252, 341, 363); and panel S (107, 117, 181, 196, 217, 247).