Amy Goshe '09 and Sasha Minium '09
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
However, bacterial resistance to antibiotic agents has risen to chaotic
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
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
operator sequence for the tetR and
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
protein, respectively. The TetA protein couples the export of
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
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
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
transcriptional regulation system extremely efficient (3).
This ensures that TetR and TetA proteins can
be synthesized before tc concentration raise to levels that inhibit
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),
antibacterial resistance remains an ongoing battle.
II. General Structure
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
protein consists of
mainly alpha-helical structure, with each monomer containing 10 alpha
helices, labeled α1- α10.
N-terminal helices α1- α3 form the DNA-binding domain while α5- α10 form the regulatory,
two domains are connected by the linker helix α4.
the DNA-binding domain, α2
form a HTH motif.
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
between the two monomers is formed by the helices α7 to α10.
portion of the regulatory domain is formed by a four-helix bundle:
at an ~80˚ angle
The regulatory domain can be divided into a rigid scaffold
subdomain and a conformational
depending on whether the region undergoes a change in conformation upon
III. Operator Sequence Binding
The major groove of
the tetO is
selectively recognized by TetR by both sequence-specific and
recognition helix, α3,
of the HTH motif,
lies perpendicular to the longitudinal axis of the operator DNA and
parallel to the major groove.
helix docking has an almost perfect orentation. In
is the main contributor in sequence-specific recognition of tetO. All
members of the helix (which include Gln
43, and His
44) aid in this recognition except for Leu
Instead of binding to DNA, Leu
41 interacts with nearby
hydrophobic residues to stabilize the HTH motif.
bonding plays a significant role in operator binding. Residues
44, Thr 40, Tyr
42, Pro 39
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.
hyrdophobic bonding between the protein and operator is
hydrogen bonds are also
hydrogen bonds with an adenine residue, Thr 40 directly contacts a thymine and
a cytosine residue,
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.
hydrogen bonds are formed on either
phosphate group closest to guanine at position +2, both with side
42, and Lys
with the amino groups of the main chains (Thr
and Lys 48).
Due to this high extent of hyrogen bonding, the nucleic acid
is pulled closer to the TetR protein near G +2.
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.
enters the cell and
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.
binds to TetR in a binding
pocket formed by helices α4, α5, α6,
. The binding pocket is composed of primarily
amino acid residues. Ring A of [MgTc]+ faces the inside of the binding
because Ring A contains the most potential hydrogen bond donors and
Due to this chemical makeup, Ring A makes anchoring hydrogen bonds with
residues His 64
to see a different view of the binding pocket.
ion (not shown) also makes contacts with the binding pocket,
specifically residues His
and Thr 103,
forcing helix α6
(the helix these two residues are attached to) to make
a 1.5 Angstrom shift in the
direction (toward α4).
change completes the formation of the
Change in the
induced complex undergoes a
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
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.
conformational change occurs as a result of the van der Waals
contact between helix
α6 (the helix
bound to the magnesium ions) and
(the linker helix connecting the DNA binding region to the
the protein core).
makes the 1.5 Å
shift to form the inducer (see previous
section), the α4
linker must shift in the
a fixed position (due to the contact between tetracycline Ring A and His
the binding pocket) and cannot move, but the α4
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.
shift of α4
TetR’s DNA binding domain, particulary at helices α2 and α3
and TetO, reducing
affinity for the operator sequence. TetR and TetO eventually
with the operator sequence no longer bound by the repressor,
the proteins TetA and TetR can occur.
system has been adapted as a transcriptional
regulator in eukaryotic cell lines, including Saccharomyces
cerevisiae and Dictyostelium
The operator sequence is inserted into the
close to the start sequence of a target gene (the most commonly studied
the RNA polymerase promoters), and in the presence of TetR,
the target gene can be significantly repressed. The best results occur
TATA box is flanked by two tetO
sequences, as all transcriptional
the target gene is repressed (6). Because of its success in regulating
transcription, the addition of a TetR-tetO system is a
in the genomes
transgenic mice and other organisms (6, 7). The TetR-tetO system allows
for greater regulatory control of isolated target genes within the
have also revealed the existence of a reverse
system. Reverse TetR is mutated in such a way that binding to
operator sequence is only possible when tetracycline or a tetracycline
is already bound (7). There is no creation
of an inducer complex required in the reverse TetR system. Click here to see a
complex. Reverse TetR
also been applied to eukaryotic transcriptional regulation and is also
used to create transgenic organisms.
Neu, H.C. 1992. The crisis in antibiotic resistance. Science
Speer, B.S., N.B. Shoemaker, & A.A. Salyers. 1992.
resistance to tetracycline: mechanisms, transfer, and
D. Schnappinger, W.
Hillen, W. Saenger, & W. Hinrichs. 2000. Structural basis of
regulation by the tetracycline inducible Tet repressor-operator system.
Wenzel, R., G. Bate, & P. Kirkpatrick. 2005.
(5) Ramos, J.L. et al.
2005. The TetR family of transcriptional
(6) Saenger, W., P. Orth, C. Kisker, W. Hillen,
& W. Hinrichs. 2000. The Tetracycline
for a Biological Switch. Angew
Chem Int Ed Engl 39(12):2042-2052.
M. & H. Bujard. 2002. Studying gene function in eukaryotes by
conditional gene inactivation. Annual
Review of Genetics 36:153-173.