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Tetracycline Repressor Complex

Amy Goshe '09 and Sasha Minium '09


I. Introduction

There exist countless antibiotics with many target different regions in bacterial cells.  Click here for a sample of the target sites (1).  For example, tetracycline (tc) binds to the 30S ribosomal subunit and inhibits protein synthesis by preventing peptide chain elongation.  Click here to see a tetracycline molecule.

However, bacterial resistance to antibiotic agents has risen to chaotic levels.  Resistance ensues through chromosomal mutation or by the exchange of DNA.  This DNA exchange can occur through conjugative plasmids or through transposons, which can enter either plasmids or chromosomes.  Resistance to tc in Escherichia coli is carried on the transposon Tn10 contained in a plasmid (2).

The main mechanism of resistance to tc in gram-negative bacteria is the efflux of the drug from the cell (3).  In resistant cells, the Tetracycline repressor protein (TetR) binds to the tetO operator sequence for the tetR and tetA genes.  The operator overlaps the promoters for these genes, thus transcription is inhibited when the repressor protein is bound.  When it diffuses into the cell, tc chelates the divalent magnesium cation to form the biologically active [MgTc]+ complex.  Then
[MgTc]+ binds to TetR, inducing an allosteric conformational change in the repressor protein that releases it from DNA.  This event allows for transcription of tetR and tetA genes, which code for the TetR protein and the TetA antiporter membrane protein, respectively.  The TetA protein couples the export of [MgTc]+ from the cell with the import of H+.  TetA is not usually synthesized since it hinders electrical potential maintenance of the cellular membrane.  The rise of TetA and TetR in the cell quickly decreases cytoplasmic levels of tc, thus restoring repression of the tetA and tetR genes.

The key to this resistance mechanism is the high affinity of
[MgTc]+ for TetR.  The association constant of [MgTc]+ binding to TetR is ~109 M-1, but that for [MgTc]+ binding to the ribosome is ~106 M-1 (3).  Furthermore, the affinity of TetR for tetO is reduced by 9 orders of magnitude upon binding of [MgTc]+ to the protein, making the TetR-tetO transcriptional regulation system extremely efficient (3).  This ensures that TetR and TetA proteins can be synthesized before tc concentration raise to levels that inhibit protein synthesis.

In the 1950s and 1960s, tc was abundantly distributed in the United States (2).  It was a desirable antibiotic due to its low toxicity, broad spectrum of activity, and relatively cheap production.  The spread of tc resistance in pathogenic bacteria limits the current clinical use of tc.  While the glycyclines have been synthesized and approved by the FDA as a second generation tc (4), fighting antibacterial resistance remains an ongoing battle.

II. General Structure

Microbial transcription factors tend to contain two domains:  a regulatory domain and a DNA-binding domain.  Approximately 95% of prokaryotic transcription factors bind DNA by adopting a helix-turn-helix (HTH) motif (5).  The TetR protein exists in bacteria as a homodimer with twofold symmetry.   The protein consists of mainly alpha-helical structure, with each monomer containing 10 alpha helices, labeled α1- α10.  The N-terminal helices α1- α3 form the DNA-binding domain while α5- α10 form the regulatory, signal-receiving domain.  These two domains are connected by the linker helix α4.

Within the DNA-binding domain, α2 and α3 form a HTH motif.  The HTH selectively binds the two adjacent major grooves that form the tetO operator.  This sequence has internal palindromic symmetry with a central base pair. Click here to see a diagram of the operator.

The dimerization surface between the two monomers is formed by the helices α7 to α10.  The core portion of the regulatory domain is formed by a four-helix bundle:  anitparallel helices α8 and α10 intersect at an ~80˚ angle with α'8 and α'10.

The regulatory domain can be divided into a rigid scaffold subdomain and a conformational change subdomain, depending on whether the region undergoes a change in conformation upon tc binding.  

III. Operator Sequence Binding

The major groove of the tetO is selectively recognized by TetR by both sequence-specific and sequence-independent interactions.

The recognition helix, α3, of the HTH motif, lies perpendicular to the longitudinal axis of the operator DNA and parallel to the major groove.  This helix docking has an almost perfect orentation.  In addition, α3 is the main contributor in sequence-specific recognition of tetO.  All members of the helix (which include Gln 38, Pro 39, Thr 40, Leu 41, Tyr 42, Trp 43, and His 44) aid in this recognition except for Leu 41.   Instead of binding to DNA, Leu 41 interacts with nearby hydrophobic residues to stabilize the HTH motif.

Hydrophobic bonding plays a significant role in operator binding.  Residues Trp 43, His 44Thr 40Tyr 42Pro 39 contribute enthalpically favorable van der Waals contacts with the DNA double helix.  Since water forms ordered structures around hyrophobic molecules, the exclusion of water from the DNA-protein interface results in a favorable increase in entropy.  Therefore, hyrdophobic bonding between the protein and operator is exergonic.  

Sequence-specific hydrogen bonds are also important.  Gln 38 hydrogen bonds with an adenine residueThr 40 directly contacts a thymine and a cytosine residue, Pro 39 interacts with a A-T base pair, along with another thymine residue, and Arg 28 hydrogen bonds with the guanine residue in the +2 position.

Sequence-independent hydrogen bonds are formed on either phosphate group closest to guanine at position +2, both with side chains (Thr 26, Thr 27, Tyr 42, and Lys 48) and with the amino groups of the main chains (Thr 26 and Lys 48).  Due to this high extent of hyrogen bonding, the nucleic acid is pulled closer to the TetR protein near G +2.  This results in a kink in the DNA away from TetR at this location. The kink is stabilized by bending in the neighboring DNA.

All base pairs of the operator are involved in binding to TetR except for the central base, which ensures proper spacing between the two halves of the operator. Click here to see another representation of the TetR-tetO interface.

IV. Formation of the Inducer Complex

Tetracycline enters the cell and binds to magnesium ions. This Tetracycline-magnesium complex (abbreviated as [Mg-Tc]+) can then bind to the repressor protein (TetR). When [Mg-Tc]+ binds the repressor protein, the inducer complex is formed. 

[MgTc]+ binds to TetR in a binding pocket formed by helices α4, α5, α6, and α7 . The binding pocket is composed of primarily hydrophobic amino acid residues. Ring A of [MgTc]+ faces the inside of the binding pocket, because Ring A contains the most potential hydrogen bond donors and acceptors. Due to this chemical makeup, Ring A makes anchoring hydrogen bonds with residues His 64 (α4), Asn 82, Phe 86 (α5), and Gln 116 (α7). Click here to see a different view of the binding pocket.

The attached magnesium ion (not shown) also makes contacts with the binding pocket, specifically residues His 100 and Thr 103, forcing helix α6 (the helix these two residues are attached to) to make a 1.5 Angstrom shift in the C-terminus direction (toward α4).

This conformational change completes the formation of the inducer complex.

V. Conformational Change in the Inducer

The induced complex undergoes a conformational change which allows the repressor to disengage from the operator sequence. This conformational change reduces the the affinity of the repressor protein for the operator sequence. When the repressor disengages, the operator is free to begin transcribing the tetR and tetA genes, which result in the production of membrane proteins that expel  tetracycline from the cell before it can bind to and interfere with the ribosome. 

The conformational change occurs as a result of  the van der Waals contact between helix α6 (the helix bound to the magnesium ions) and helix α4 (the linker helix connecting the DNA binding region to the rest of the protein core). When α6 makes  the 1.5 Å shift to form the inducer (see previous section), the α4 linker must shift in the same direction. α4’s C-terminus is in a fixed position (due to the contact between tetracycline Ring A and His 64 in the binding pocket) and cannot move, but the α4 N-terminus can. The N-terminus swings in a pendulum-like motion approximately 5 degrees inward toward the protein core of TetR. Click here to see a different view of the conformational change. 

The pendulum shift of α4 alters the contact between TetR’s DNA binding domain, particulary at helices α2 and α3 , and TetO, reducing TetR’s binding affinity for the operator sequence. TetR and TetO eventually dissociate, and with the operator sequence no longer bound by the repressor, transcription of the proteins TetA and TetR can occur. 

VI. Applications

The TetR-tetO system has been adapted as a transcriptional regulator in eukaryotic cell lines, including Saccharomyces cerevisiae and Dictyostelium discoideum. The operator sequence is inserted into the eukaryotic genome close to the start sequence of a target gene (the most commonly studied being the RNA polymerase promoters), and in the presence of TetR, transcription of the target gene can be significantly repressed. The best results occur when the TATA box is flanked by two tetO sequences, as all transcriptional activity of the target gene is repressed (6). Because of its success in regulating eukaryotic transcription, the addition of a TetR-tetO system is a common mutation in the genomes of transgenic mice and other organisms (6, 7). The TetR-tetO system allows for greater regulatory control of isolated target genes within the trangenic organism.

Mutant screens have also revealed the existence of a reverse TetR-tetO system. Reverse TetR is mutated in such a way that binding to the operator sequence is only possible when tetracycline or a tetracycline analog is already bound (7).  There is no creation of an inducer complex required in the reverse TetR system. Click here to see a reverse TetR complex. Reverse TetR has also been applied to eukaryotic transcriptional regulation and is also used to create transgenic organisms.

VII. References

(1) Neu, H.C. 1992. The crisis in antibiotic resistance. Science 256:1063-1074.

(2) Speer, B.S., N.B. Shoemaker, & A.A. Salyers.  1992.  Bacterial resistance to tetracycline:  mechanisms, transfer, and clinical signficiance. CMR 5(4):387-399.

(3) Orth, Peter, D. Schnappinger, W. Hillen, W. Saenger, & W. Hinrichs. 2000. Structural basis of gene regulation by the tetracycline inducible Tet repressor-operator system. Nature Structural Biology 7(3):215-219.

(4) Wenzel, R., G. Bate, & P. Kirkpatrick.  2005.  Tigecycline.  NPG 4(10):809-810.

(5) Ramos, J.L. et al.  2005.  The TetR family of transcriptional repression.  MMBR 69(2):326-356.

(6) Saenger, W., P. Orth, C. Kisker, W. Hillen, & W. Hinrichs. 2000. The Tetracycline Repressor-A Paradigm for a Biological Switch. Angew Chem Int Ed Engl 39(12):2042-2052.

(7) Gossen, M. & H. Bujard. 2002. Studying gene function in eukaryotes by conditional gene inactivation. Annual Review of Genetics 36:153-173.

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