Download Programming Gene Expression

Dr. Muhammad Zeeshan Hyder
Department of Biosciences CIIT Islamabad
Chapter 31, BIOCHEMISTRY, by Lubert Stryer 5th Edition
Programming Gene Expression
Complex biological processes often involve coordinated control of the expression of many genes. The maturation of a
tadpole into a frog is largely controlled by thyroid hormone. This hormone regulates gene expression by binding to a protein, the
thyroid hormone receptor, shown at the right. In response to hormone binding, this protein binds to specific DNA sites in the
genome and modulates the expression of nearby genes. [(Left) Shanon Cummings/Dembinsky Photo Associates.]
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Bacteria are highly versatile and responsive organisms: the rate
of synthesis of some proteins in bacteria may vary more than a
1000-fold in response to the supply of nutrients or to
environmental challenges.
Cells of multicellular organisms also respond to varying
conditions. Such cells exposed to hormones and growth factors
will change substantially in shape, growth rate, and other
characteristics.
Many different cell types are present in multicellular organisms. For example, cells from muscle and nerve tissue show
strikingly different morphologies and other properties, yet they contain exactly the same DNA. These diverse properties are
the result of differences in gene expression. A comparison of the gene-expression patterns of cells from the pancreas, which
secretes digestive enzymes, and the liver, the site of lipid transport and energy transduction, reveals marked differences in
the genes that are highly expressed, a difference consistent with the physiological roles of these tissues.
Gene expression is the combined process of the transcription of a gene into mRNA, the processing of that mRNA, and its
translation into protein (for protein-encoding genes).
It is controlled first and foremost at the level of transcription. Much of this control is achieved through the interplay
between proteins that bind to specific DNA sequences and their DNA binding sites.
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Most of the genes in prokaryotic genomes are arranged in operons. Which
are contains genes for a related biochemical pathway or help in adaptation to
a new change in environment in a coordinated way.
Lac operons help bacteria to adjust to lactose in absence of glucose. Bacteria
such as E. coli usually rely on glucose as their source of carbon and energy.
However, when glucose is scarce, E. coli can use lactose as their carbon. An
essential enzyme in the metabolism of lactose is b-galactosidase, which
hydrolyzes lactose into galactose and glucose.
The amount of is b-galactosidase increases in response to lactose from about 10 molecules to
thousands of them by regulating the transcription of its genes.
A crucial clue to the mechanism of gene regulation was the observation that two other
proteins are synthesized in concert with b-galactosidase namely, galactoside permease and
thiogalactoside transacetylase.
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The permease is required for the transport of lactose across the bacterial cell
membrane.
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The transacetylase is not essential for lactose metabolism but appears to play a role in
the detoxification of compounds that also may be transported by the permease.
Thus, the expression levels of a set of enzymes that all contribute to the adaptation to a given
change in the environment change together. Such a coordinated unit of gene expression is
called an operon.
The coordinated expression of b-galactosidase and other genes suggested the idea of common
regulation of these gene. Francoise Jacob and Jacques Monod proposed the idea of operon model.
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The model consists of following genetic elements:
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A regulator gene ( “i gene” that encodes a repressor proteins which binds at operator site to
repress the transcription)
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a regulatory DNA sequence called an operator site (the binding site for repressor designated as
“o”)
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and a set of structural genes (“z, y and a genes” encoding b-galactosidase, the permease and
transacetylase).
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The promoter site (‘p’) is the site where RNA polymerase binds and starts the transcription process.
The structural genes are encoded by a single mRNA and this type of mRNA is said to be as polygenic or
polysictronic transcript.
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The Lac operator Has a Symmetric Base Sequence: The
sequence of operator site showed two fold symmetry which
help repressor protein to bind at this site as dimmer.
Symmetry matching is a recurring theme in protein-DNA
interactions
The lac repressor can exist as a dimer of 37-kd subunits. In the
absence of lactose, the repressor binds very tightly and
rapidly to the operator and prevent RNA polymerase from
locally unwinding the DNA to synthesize of the mRNA
The lac repressor binds 4 × 106 times as strongly to operator DNA as it does to random
sites in the genome (4.6 ×106 bases genome). of other sites within.
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The dissociation constant for the repressor-operator complex is approximately 0.1
pM (10-13 M).
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The rate constant for association ( 1010 M-1 s-1) is strikingly high, indicating that the
repressor by one dimensional search!
The E. coli genome sequence has two sites within 500 bp of the primary operator site are
similar the sequence of the operator. No other sites that closely match sequence of the
lac operator site are present in the rest of the E. coli genome sequence. Thus, the DNAbinding specificity of the lac repressor is sufficient to specify a nearly unique site within
the E. coli genome.
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The ligand for lac operon is allolactose which induces the expression of lac operon: The
lactose itself not bind with the repressor rather a side product which is produced when lactose
comes into the cell by the b-glactosiddase (which is produced at low level in repressed state)
reaction binds with lac repressor protein.
The binding of the ligand (inducer) with the repressor brings local conformational changes
that are transmitted to the interface with the DNA-binding domains resultantly when the lac
repressor is bound to the inducer, the repressor's affinity for operator DNA is greatly reduced.
The processes that regulate gene expression in the lactose operon: In the absence of inducer,
the lac repressor is bound to DNA in a manner that blocks RNA polymerase from transcribing
the z, y, and a genes. Thus, very little b-galactosidase, permease, or transacetylase are
produced. The addition of lactose to the environment leads to the formation of allolactose. This
inducer binds to the lac repressor, leading to conformational changes and the release of DNA by
the lac repressor. With the operator site unoccupied, RNA polymerase can then transcribe the
other lac genes and the bacterium will produce the proteins necessary for the efficient
utilization of lactose.
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The Many other gene-regulatory networks function in ways analogous to
those of the lac operon.
For example, genes taking part in purine and, to a lesser degree,
pyrimidine biosynthesis are repressed by the pur repressor. This dimeric
protein is 31% identical in sequence with the lac repressor and has a
similar three-dimensional structure.
However, the behavior of the pur repressor is opposite that of the lac
repressor: whereas the lac repressor is released from DNA by binding to a
small molecule, the pur repressor binds DNA specifically only when bound
to a small molecule.
Such a small molecule is called a corepressor. For the pur repressor, the
corepressor can be either guanine or hypoxanthine. The dimeric pur
repressor binds to inverted repeat DNA sites of the form 5 ANGCAANCGNTTNCNT-3 , in which the bases shown in yellow are
particularly important.
Examination of the E. coli genome sequence reveals the presence of more
than 20 such sites, regulating 19 operons including more than 25 genes.
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There are also DNA-binding proteins that stimulate transcription. One particularly well studied
example is the catabolite activator protein (CAP), which is also known as the cAMP response
protein (CRP).
CAP binds with cAMP to forms a dimer. and then stimulates the transcription of lactose- and
arabinose-catabolizing genes as sequence-specific DNA-binding protein.
The E. coli genome contains many CAP-binding sites in positions appropriate for interactions with
RNA polymerase. Thus, an increase in the cAMP level inside an E. coli bacterium results in the
formation of CAP-cAMP complexes that bind to many promoters and stimulate the transcription
of genes encoding a variety of catabolic enzymes.
Glucose has inhibitory effects on these genes called catabolite repression, because it lowers the
intracellular concentration of cyclic AMP. As it would be wasteful to synthesize these enzymes
when glucose is abundant.
Within the lac operon, CAP binds to an inverted repeat near to position -61 relative to the start
site for transcription and stimulates the initiation of transcription by approximately a factor of 50.
The CAP interacts with RNA polymerase and these energetically favorable protein-protein
contacts increase the likelihood that transcription will be initiated at sites to which the CAPcAMP complex is bound.
In lac operon, gene expression is maximal when the binding of allolactose relieves the
inhibition by the lac repressor, and the CAP-cAMP complex stimulates the binding of RNA
polymerase.
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Gene regulation is significantly more complex in eukaryotes than in
prokaryotes
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Genome size is different so is the number of genes
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I.e. Yeast genome is contains 16 chromosomes ranging in size from 0.2 to
2.2 Mb with 600 genes in total of 17 Mb genome.
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Humans have 46 chromosomes of 50 to 250 Mb size range with 3.2
billion bases coding for 30,000 to 40,000 gene.
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The DNA is wrapped around special histone proteins that compacts it
tremendously.
.  Every gene in the genome is expressed separately not in the form of
operons
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The transcription and translation are not coupled removing many
potential regulatory mechanisms to operate.
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The DNA in eukaryotic chromosomes is not bare. Rather eukaryotic
DNA is tightly bound to a group of small basic proteins called histones
Basic in nature quarter of the residues in each histone is either
arginine or lysine
In 1974, Roger Kornberg proposed that chromatin is made up of
repeating units, each containing 200 bp of DNA and two copies each
of H2A, H2B, H3, and H4, called the histone octomer.
These repeating units are known as nucleosomes.
The winding of DNA around the nucleosome core contributes to
DNA's packing by decreasing its linear extent. An extended 200-bp
stretch of DNA would have a length of about 680 Å. Wrapping this
DNA around the histone octamer reduces the length to
approximately 100 Å along the long dimension of the nucleosome.
Thus the DNA is compacted by a factor of seven. However, human
chromosomes in metaphase, which are highly condensed, are
compacted by a factor of 10.
This compact structure require that genome is regulated in more
complex manner.
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Chromatin structure play a role in the control of gene expression
Early experiments to digest the DNA with DNases showed some sensitive sites which are correlated with
developmental stage of organism. Indicating the change in chromatin structure.
Enhancers Can Stimulate Transcription by Perturbing Chromatin Structure
Enhancers are DNA sequences, haveing no promoter activity of their own but greatly increase the activities
of many promoters in eukaryotes, from a distant place in the genome.
Enhancers function by serving as binding sites for specific regulatory proteins which participate in
modification of histone side chains to change the binding properties of DNA with histone.
histone acetyltransferases (HATs) transfer the acetyl group from acetyl CoA
The consequences of histone acetylation are that Lysine bears a positively charged ammonium group at
neutral pH.
The addition of an acetyl group generates an uncharged amide group. This change dramatically reduces the
affinity of the tail for DNA and modestly decreases the affinity of the entire histone complex for DNA
This results in the remodeling of chromatin at that site making DNA accessible for transcriptional factors to
recruit RNA polymerase.
Thus, histone acetylation can activate transcription through a combination of three mechanisms:
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by reducing the affinity of the histones for DNA,
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by recruiting other components of the transcriptional machinery,
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and by initiating the active remodeling of the chromatin structure.
While histone deacetylation by histone deacetylase enzymes leads to transcriptional repression
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The modification of DNA provides another mechanism.
The methylation of cytosine residues plays important role in inhibiting inappropriate
gene expression in specific cell types.
Approximately 70% of the 5 -CpG-3 sequences in mammalian genomes are methylated at
the C-5 position of cytosine by specific methyltransferases. However, the distribution of
these methylated cytosines varies, depending on the cell type and state of expression of
genes.
For example the globin genes. In cells that are actively expressing hemoglobin, the region
from approximately 1 kb upstream of the start site of the b-globin gene to approximately
100 bp downstream of the start site contains fewer 5-methylcytosine residues than does
the corresponding region in cells that do not express these genes.
The relative absence of 5-methylcytosines near the start site is referred to as
hypomethylation.
The methyl group of 5-methylcytosine protrudes into the major groove where it could
easily interfere with the binding of proteins that stimulate transcription.
Although methylation is important for gene regulation but it is not a universal regulatory
Device i.e. Drosophila DNA is not methylated at all.
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Such protein-protein interactions like CAP and RNA polymerase in prokaryotic cells, play a dominant
role in eukaryotic gene regulation.
In eukaryotes each factor recruits other proteins to build up large complexes that interact with the
transcriptional machinery to activate or repress transcription.
A major advantage of this mode of regulation is that a given regulatory protein can have different
effects, depending on what other proteins are present in the same cell. This phenomenon, called
combinatorial control, and is important for development of different cell types.
The acetylated lysine residues in histone interact with a specific acetyl lysine-binding domain like one
known as bromodomain which is present in many proteins that regulate eukaryotic transcription.
Bromodomain-containing proteins are components of two large complexes essential for
transcription.
One complex of more than 10 polypeptides that consists of:
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TATA-box-binding protein
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and Proteins that bind to the TATA-box-binding protein known as TAFs (for TATA-box-binding
protein associated factors).
This complex interact with RNA polymerase to initiate transcription.
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The other complex which contains bromodomains are also present in some
components of large complexes known as chromatin-remodeling engines.
These complexes, which also contain domains homologous to those of helicases ,
utilize the free energy of ATP hydrolysis to shift the positions of nucleosomes
along the DNA and to induce other conformational changes in chromatin .
shift the positions of nucleosomes along the DNA and to induce other
conformational changes in chromatin. Histone acetylation can lead to
reorganization of the chromatin structure, potentially exposing binding sites for
other factors
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After understanding the basic mechanisms of eukaryotic gene regulations we will focus on
two examples in details.
Steroids and Related Hydrophobic Molecules Pass Through Membranes and Bind to
DNA-Binding Receptors
Like prokaryotes, eukaryotes also response to many singles present in their environment,
like hormones in the blood.
For example estrogens which are cholesterol derived steroid hormones including estrone
and progesterone involve in the regulation of ovarian cycle.
These are hydrophobic easily diffuse across cell membranes
When inside a cell, estrogens bind to highly specific, soluble receptor proteins known as
Estrogen receptors are members nuclear hormone receptors family
These receptors contains two highly conserved domains:
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a DNA-binding domain
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and a ligand-binding domain
They bind DNA sequence specifically like estrogen binds with (estrogen response
elements or EREs) of 5 -AGGTCANNNTGACCT-3 consensus sequence as dimer.
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Nuclear Hormone Receptors Regulate Transcription by
Recruiting Coactivators and Corepressors to the
Transcription Complex.
The ligand binding domain of Estrogen nuclear receptors
recruits coactivator proteins such as SRC-1 (steroid receptor
coactivator-1), GRIP-1 (glucocorticoid receptor interacting
protein-1), and NcoA-1 (nuclear hormone receptor
coactivator-1).
These coactivators inturns recruits DNA remodeling proteins
which relaxes the DNA and making a transcriptional complex,
thus inducing the transcription.
Corepressor suppress the transcription
in the similar way sequence specifically
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Nuclear Hormone Receptors Regulate Transcription by Recruiting Coactivators
and Corepressors to the Transcription Complex.
Some signaling molecules donot enter into the cell rather binds to receptors in the cell
membrane.
Like the binding of epinephrine to a 7TM receptor results in the activation of a G protein.
The activated G protein, in turn, binds to and activates adenylate cyclase, increasing the
intracellular concentration of cAMP.
This cAMP binds to the regulatory subunit of protein kinase A (PKA), activating the enzyme.
PKA also phosphorylates the cyclic AMP-response element binding protein (CREB), a
transcription factor that binds specific DNA sequences as a dimer.
The phosphorylated CREB binds a coactivator protein termed CBP, for CREB-binding protein,
which is a coactivator
This proteins recruits DNA remodeling and transcriptional complex proteins to start
transcription.
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The regulation of gene expression also takes place after the transcription in prokaryotes and
eukaryotes.
Attenuation Is a Prokaryotic Mechanism for Regulating Transcription Through Modulation
of Nascent RNA Secondary Structure
The tryptophan operon has 7-kb mRNA transcript which encodes five enzymes that convert
chorismate into tryptophan.
The mode of regulation of this operon is called attenuation, and it depends on features at
the 5 end of the mRNA product.
Upstream of the coding regions for the enzymes responsible for tryptophan biosynthesis
lies a short open reading frame encoding a 14-amino-acid leader peptide.
Following this open reading frame is a region of RNA, called an attenuator, that is capable
of forming several alternative structures.
The translation of the trp mRNA begins soon after the ribosome-binding site has been
synthesized.
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A ribosome is able to translate the leader region of the mRNA product only in the presence of
adequate concentrations of tryptophan.
When enough tryptophan is present, a stem-loop structure forms in the attenuator region,
which leads to the release of RNA polymerase from the DNA.
However, when tryptophan is scarce, transcription is terminated less frequently due to the
formation of another secondary structure as ribosomes do not stalls over their to wait for
tryptophanyl-tRNA, which is the case when tryptophan is less.
Many other amino acid synthesis operons are regulated in this way.
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RNA secondary structure plays a role in the regulation of iron
metabolism in eukaryotes.
Iron is an essential nutrient, but excess iron can be quite
harmful because, untamed by a suitable protein environment,
iron can initiate a range of free-radical reactions that damage
proteins, lipids, and nucleic acids.
Animals have evolved sophisticated systems for iron safe
storage .
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Transferrin, a transport protein that carries iron in the
serum,
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Transferrin receptor, a membrane protein that binds ironloaded transferrin and initiates its entry into cells, and
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Ferritin, an impressively efficient iron-storage protein
found primarily in the liver and kidneys.
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Twenty-four ferritin polypeptides form a nearly spherical
shell that encloses as many as 2400 iron atoms, a ratio of
one iron atom per amino acid
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Ferritin and transferrin receptor expression levels are
reciprocally related in their responses to changes in iron
levels.
When iron is scarce, the amount of transferrin receptor
increases and little or no new ferritin is synthesized.
This regulation takes place at the level of translation.
Ferritin mRNA includes a stem-loop structure termed an
iron-response element (IRE) in its 5’ untranslated region.
This stem-loop binds a 90-kd protein, called an IREbinding protein (IRE-BP), that blocks the initiation of
translation.
When the iron level increases, the IRE-BP binds iron as a
4Fe-4S cluster. The IRE-BP bound to iron cannot bind RNA,
because the binding sites for iron and RNA substantially
overlap. Thus, in the presence of iron, ferritin mRNA is
released from the IRE-BP and translated to produce
ferritin, which sequesters the excess iron.
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The transferrin-receptor mRNA contains many IRE-like
regions. However, these regions are located in the 3’
untranslated region rather than in the 5 untranslated
region.
Under low-iron conditions, IRE-BP binds to these IREs
but still these mRNA are translated due IRE position at
3’ end.
When the iron level increases and the IRE-BP no longer
binds transferrin-receptor mRNA.
Freed from the IRE-BP, transferrin-receptor mRNA is
rapidly degraded.
Thus, an increase in the cellular iron level leads to the
destruction of transferrin-receptor mRNA and, hence, a
reduction in the production of transferrin-receptor
protein.
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