Tetracycline
Repressor Complex
Amy Goshe '09 and Sasha Minium '09
Contents:
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
44, Thr 40, Tyr
42, Pro 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 residue, Thr 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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