Download Genetic Switches - 1

NPTEL – Biotechnology - Systems Biology
Genetic Switches - 1
Dr. M. Vijayalakshmi
School of Chemical and Biotechnology
SASTRA University
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Table of Contents
1 INTRODUCTION .............................................................................................. 3
1.1 TYPICAL GENE SWITCHING SYSTEMS IN CELLS ................................................ 4
2 BACTERIAL RESPONSE TO A SINGLE SIGNAL- THE ................................. 5
TRYPTOPHAN REPRESSOR AS A BACTERIAL SWITCH ............................ 5
2.1 ATTENUATION ................................................................................................ 7
3 REFERENCES .................................................................................................. 8
3.1 TEXT BOOKS .................................................................................................. 8
3.2 LITERATURE REFERENCES .............................................................................. 8
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1 Introduction
This is the right time to discuss an interesting experiment designed by Andr’e
Lwoff, Francis Jacob and Jacques Monod at the Pasteur institute in Paris nearly
5 decades back. Their experiment showed that a strain of the bacterium E.coli
irradiated with ultraviolet light halted their growth and after nearly 90 minutes
lyses releasing a crop of viruses into the culture medium. These viruses
(originally called lambda) are also called bacteriophages. In many bacteria the
lambda virus is dormant but several other bacteria infected by the virus lyse,
producing new phage. The normal growth and division when repeated produces
a crop of new phages. This experiment clearly demonstrated that the virus
switches between two states from the dormant state in the dividing bacterium to
the activated state in the bacterium irradiated with UV light. This is a striking
example of turning ‘ON’ or ‘OFF’ specific genes. We know that genes are the
functional components of a living cell. Such living cells (bacterial or human)
utilise only a subset of their genes to signal the production of other molecules.
Therefore those genes which are expressed are termed ‘ON’ and those not
expressed are turned ‘OFF’. In other words we call this phenomenon regulation
of gene expression.
This regulation of gene expression as an event takes place not only during the
developmental cycle but throughout the life time of a diffrentiative cell.
Gene regulatory proteins and the specific sequences of DNA recognised by
these proteins form the basic components of ‘genetic switches’. It has been
shown that nearly 80% of genetic material of gene switches alters the function of
a gene. These genetic switches could be activated or deactivated by external
signals, toxins, medium deprivation, stress etc. For example, continuous
exposure to sunlight changes the colour of skin cells. Sunlight does not change
the structure of the pigmentation gene but alters its function by turning the gene
‘ON’. The brilliant discovery of gene switching ON and OFF in lambda phage
revolutionised several new experiments in the field and established that the
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switching mechanism observed in E.coli and lambda phage also applied to
eukaryotic cells.
1.1 Typical Gene Switching Systems in Cells
Skeletal muscle is composed of two types of fibers- slow twitch fibers which are
innately vascular and fast twitch fibres which are deficient in blood vessels. In
one of the clinical complications called Critical limb ischemia, blood flow to
skeletal muscle is blocked leading to muscle wasting and eventually to the
amputation of the limbs. Experiments have established that the genetic switch
estrogen related receptor gamma (ERR gamma) when expressed in fast twitch
fibers converts them into slow twitch fibers resulting in a significant increase in
blood supply to the skeletal muscle. This is a classic case of treating a clinical
complication without a pharmacological intervention.
TORC2 is a protein which promotes gluconeogenesis in liver under hypoglycemic
conditions by functioning as a metabolic switch. This protein resides outside the
nucleus under normal conditions but upon oxidative stress or starvation, shuttles
to nucleus and activates a network of genes, vital for handling the insult.
Mutations in the TORC protein have been shown to reduce the life expectancy.
TORC mutated flies lose their lipid storing capability and have been shown to
regain starvation and stress resistance when TORC is expressed in the nervous
system.
Experiments on hypoxia tolerant fruit flies have shown that hairy, a transcriptional
suppressor is critical for cell survival under hypoxic conditions. Hairy gene may
shut off or hinder activation of many genes. On activation it restrains various
signalling pathways allowing the cells to circumvent hypoxia. It activates a sort of
cutback mechanism in cells culminating in energy conservation which is then
used for important functions. Let us now, discuss genetic switches in bacteria.
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2 Bacterial Response to a Single Signal- The
Tryptophan repressor as a Bacterial Switch
The genome of E.coli encodes approximately 4,200 proteins. Its chromosome
comprises 4.6x106bp. The expression of many of the genes in E.coli is heavily
dependent on the availability of nutrients in the environment.
Tryptophan is a rare amino acid and is a precursor for niacin in eukaryotes. In
bacteria, indole is formed from tryptophan. In plants it acts as precursor for
biosynthesis the plant hormone auxins. Tryptophan is synthesized from
chorismate in five steps catalyzed by three different enzymes which are
produced by 5 genes as in Fig 1.The five genes include TrpE, TrpD, TrpC, TrpB,
TrpA. Upstream to TrpE lies TrpL, operator, promoter and far from this stretch
lies TrpR. These genes are arranged adjacent to each other on the chromosome
as a single operon. The five genes are transcribed as a single mRNA molecule
from a single promoter. When tryptophan in the growth medium enters the cell,
the cell does not require these enzymes and therefore shuts off the production of
these enzymes. The molecular mechanism of the tryptophan switch has been
clearly understood and established. The tryptophan repressor is a member of
the Helix-Turn-Helix family in which the promoter and the operator are arranged
to facilitate classic switching.
Fig 1. Gene regulation in Trp operon. When the level of trptophan is high: Repression occurs in the circuit.
Lower levels of tryptophan results in expression of a gene
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In the first step, chorismate is converted to anthranilate. Anthranilate is converted
into phosphoribosylanthranilate which is then converted into carboxy phenyl
aminodeoxy ribulose -5-phosphate. After this indole- 3 –glycerol phosphate is
formed which is ultimately converted into L-tryptophan via indole formation.
TrpL is a leader sequence and contains a critical region called TrpA meant for
attenuation. TrpR produces inactive repressor called apo repressor which can’t
bind the operator tryptophan acts as corepresssor and activates apo repressor
for shutting down genes. The repressor and apo repressor are dimers made up
of identical Helix-Turn-Helix monomers. Tryptophan binding prepares the
repressor for precise interaction with operator sequences. Trp repressor weakly
regulates its own synthesis by binding with an operator site located in its
promoter.
In a tryptophan switch, gene expression is regulated through a novel but simple
mechanism. In order to bind to the operator DNA, the repressor protein should
bind to the amino acid tryptophan through two of its molecules. The binding of
tryptophan realigns the Helix-Turn-Helix motif of the repressor presenting it to the
major groove of the DNA. When tryptophan is not present, the motif swings
inward preventing the binding of the protein to the operator. The tryptophan
repressor and the operator thus form an elegant switching device that turns gene
ON and OFF.
Tryptophan repressor is an example of a negative repressible operon. (Negative
with reference to repressor and repressible with reference to tryptophan). If the
tryptophan is meager, bacterial structural genes necessary for converting
chorismate to tryptophan are activated as the repressor made by TrpR is inactive
and is unable to bind to the operator.
In the presence of tryptophan, structural genes need not transcribe as this may
result in energy wastage. Tryptophan binds the inactive repressor making it
functional .The active repressor binds to operator providing hindrance for RNA
polymerase binding. This results in gene repression.
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2.1 Attenuation
Premature termination of primary transcript in the leader region i.e. before the
first structural genes is called attenuation. Attenuation is carried out by
attenuator, a sequence within leader region of the tryptophan operon, (Fig 2). At
this site, choice is made by RNA polymerase either to terminate or continue
transcription. Mutants with small deletions in this region produce tryptophan
synthesizing enzymes even in the presence of tryptophan.
Fig 2. Termination of Transcription regulated by attenuation
(a) Stem-loop structures of the trp operon in the mRNA;
(b) Low level of trp full length mRNA made;
(c) High level transcription of the trp operon is prematurely halted
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3 References
3.1 Text Books
1. Alberts B. Bray D, Lewis J. et al., Molecular Biology of the Cell, Garland
Science, (1994)
2. Mark Ptashne, A Genetic Switch-Phage Lambda Revisited, CSHL Press,
U.S.A, (2004).
3.2 Literature References
1. Gardner Timothy S et al., Construction of a genetic toggle switch in
Escherichia coli, Nature, (2000), 403, 339-342.
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