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Regulation of Gene
Expression
基因表达调控
Deqiao Sheng PhD
Dept. of Biochemistry and
Molecular Biology
Reference Books
1.
2.
3.
4.
Leningers’ Principles of Biochemistry
Harpers’ Biochemistry 26th edition
Styers’ Biochemistry
Hortons’ Principles of Biochemistry 4th
edition
Basic conceptions
Diagram of the central
dogma, DNA to RNA to
protein, illustrating the
genetic code.
Gene expression

A gene(基因) is the basic unit of heredity in
a living organism.
All living things depend on genes. Genes hold
the information to build and maintain an
organism's cells and pass genetic traits to
offspring.
 Function units


Genome——is the entirety of an organism's
hereditary information. It is encoded either in
DNA or, for many types of virus, in RNA.


E.coli contains about 4,400 genes present on a single
chromosome
Human genome is more complex, with 23 pairs of
chromosomes containing 6 billion(6×109) base pairs of
DNA. 30,000~40,000 genes
Concept of gene expression

Gene expression is the combined process of the
transcription of a gene into mRNA, the processing
of that mRNA, and its translation into protein

Gene expression is the process by which
information from a gene is used in the synthesis of
a functional gene product. These products are
often proteins, but in non-protein coding genes
such as rRNA genes or tRNA genes, the product is
a functional RNA.

The genetic information present in each
somatic cell of a metazoan organism
(multicellular animals ) is practically
identical.
How to meet the different needs?
Different function need different proteins.

Regulated expression of genes is required
for development, differentiation, and
adaption.
In genetics gene expression is the most
fundamental level at which genotype gives
rise to the phenotype.
 The genetic code is "interpreted" by gene
expression, and the properties of the
expression products give rise to the
organism's phenotype.
 Genotype→Phenotype

Genotype
Information Flow

A gene is turned on and
transcribed into RNA
 Information flows from
genes to proteins,
genotype to phenotype
Phenotype
The cellular
concentration of a
protein is
determined by a
delicate balance of
at least seven
processes, each
having several
potential points of
regulation.
Points of Regulation
1.
2.
3.
4.
5.
6.
7.
Transcription
Post-transcriptional modification
mRNA degradation rate
Translation
Post-translational modification
Protein targeting and transport
Protein degradation
Regulation of Gene Expression
General
 The regulation of the expression of genes is
absolutely essential for the growth,
development, differentiation and the very
existence of an organism.
 The are two types of gene regulationpositive and negative.
•
•
Positive regulation: the gene regulation is
said to be positive when its expression is
increased by a regulatory element (positive
regulator)
Negative regulation: A decrease in the
gene expression due to the presence of a
regulatory element (negative regulator) is
referred to as negative regulation.
The aim of the control
What
When
Where
To select the right gene
To express at the right time
To express at the right place
The right gene expresses at the right time
& the right place.
The significance of gene
expression regulation


The differential transcription of different genes
largely determines the actions and properties of
cells.
Regulation at any one of the various steps in this
process could lead to differential gene expression
in different cell types or developmental stages or
in response to external conditions (such as:
Environments).
 Temporal specificity (stage specificity)
 Spatial specificity (cell or tissue specificity)
FROM EGG TO ORGANISM:
HOW AND WHY GENES ARE REGULATED
• Four of the many
different types of
human cells
– They all share the
same genome
Genotype (DNA)
– What makes them
different?
Phenotype (Protein)
One of underlying principles of
molecular cell biology is that the
actions and properties of each cell
type are determined by the proteins it
contains.
(a) Three muscle cells (partial)
(b) A nerve cell (partial)
(c) Sperm cells
(d) Blood cells
One of the characters of gene expression : it is
precisely controlled to be activated in the right
cells and right time during development of the
many different cell types that collectively form a
multicellular organism.
e.g. Human Hemoglobin(血红蛋白)
• Human hemoglobin is consisted of two alpha-like
and beta-like globin chains, which are coded by
alpha-like and beta-like globin genes respectively.
Hemoglobin
clusters
Human hemoglobin: (at developmental stages)
z 2e 2
HbF a2g2 (end of trimester)
HbA a2b2 (start from the third trimester ,
do not completely replace g chains until some
weeks postpartum)
Regulation of Gene Expression
1.
2.
3.
Principles of gene regulation
Regulation of gene expression in
prokaryotes
Regulation of gene expression in
eukaryotes
Principles of Gene Regulation
Principles of Gene Regulation
1.
Constitutive gene expression

2.
A gene is expressed at the same level at all
times.
housekeeping gene
Regulated gene expression


Inducible :Gene products that increase in
concentration under particular molecular
circumstances.
Repressible: gene products that decrease
in concentration in response to a
molecular signal.
Constitutive genes

Constitutive genes: refer to genes whose
expression are not regulated. The products of
these genes are produced at a constant rate. Such
genes are called constitutive genes and their
expression is said to be constitutive. e.g.
b-actin-Actins are highly conserved proteins that are
involved in cell motility, structure and integrity.
GAPDH (glyceraldehyde-3-phosphate
dehydrogenase )-is an enzyme that catalyzes the
sixth step of glycolysis and thus serves to break down
glucose for energy and carbon molecules.

Products of the constitutive genes are
required at all times, such as those for the
enzymes of central metabolic pathways.
Those genes are expressed at a more or less
constant level in virtually every cell of a
species or organism. They are often
referred to as housekeeping genes also.
Inducible genes

Inducible genes refer to the genes whose
expression increases in response to an
inducer, a specific regulatory signal. The
process is called induction.
e.g.
The expression of many of the genes encoding
DNA repair enzymes, for example, is induced by
high levels of DNA damage.
The Structure of Gene

Structural gene


codes for a protein (or RNA) product
Regulatory gene

codes for a protein (or an RNA) involved in
regulating the expression of other genes
Structural and Regulatory gene
A structural
gene :
Structural genes represent an enormous variety of
protein structures and functions, including
structural proteins, enzymes and regulatory
proteins.
A regulatory
gene :
The interaction can regulate a target gene in a
manner either positive (the interaction turns the
gene on) or negative (the interaction turns the
gene off).
RNA Polymerase Binds to DNA
at Promoters


RNA polymerases bind to DNA and initiate
transcription at promoters , sites generally found
near points at which RNA synthesis begins on the
DNA template.
The nucleotide sequences of promoters vary
considerably, affecting the binding affinity of RNA
polymerases and thus the frequency of
transcription initiation.
Sequences of promoters
Consensus sequence for many E. coli
promoters. (procaryotic)
-10 region TATAAT
-35 region TTGACA
Most base substitutions in the -10 and -35
regions have a negative effect on promoter
function. Some promoters also include the
UP (upstream promoter) element
Transcription activity ?
 promoter
sequence
 Mutations
that result in a shift away from
the consensus sequence usually decrease
promoter function; conversely, mutations
toward consensus usually enhance
promoter function.
 regulatory
 It
proteins
can modulate non-housekeeping genes
expression
Common sequences in promoters recognized by
eukaryotic RNA polymerase II.
 -30 region TATA box
 Initiator sequence (Inr)
N , represents any nucleotide
Y, a pyrimidine nucleotide
RNA Polymerase II Requires Many
Other Protein Factors for Its Activity
1. specificity factors
–
Alter the specificity of RNA polymerase for a
given promoter or set of promoters
2. repressors
–
impede access of RNA polymerase to the
promoter
3. activators
–
Enhance the RNA polymerase–promoter
interaction.
RNA polymerase II
holoenzyme complex
bound to a promoter
Transcription
machinery

There are a lot of proteins participate in the
regulation of gene expression.
–
–
–
–
Transcription Factor (TF)
Activators
Repressors
Regulatory proteins
Specificity factors

Prokaryotic specificity factors
– The  subunit of the E. coli RNA polymerase
holoenzyme is a specificity factor that mediates
promoter recognition and binding.

Eukaryotic specificity factors
– the TATA-binding protein (TBP)
Repressors

Protein
 Bind to specific sites on the DNA
In prokaryotic cells, such binding sites,
called operators, are generally near a
promoter.
 Blocks transcription/negative regulation
Activators

Activators provide a molecular
counterpoint to repressors; they bind to
DNA and enhance the activity of RNA
polymerase at a promoter
 positive regulation
 binding sites are often adjacent to
promoters that are bound weakly or not
at all by RNA polymerase alone
Enhancers

positive regulation
Some eukaryotic activators bind to DNA sites,
called enhancers, that are quite distant from the
promoter, affecting the rate of transcription at a
promoter that may be located thousands of base
pairs away.
 Some activators are normally bound to DNA,
enhancing transcription until dissociation of the
activator is triggered by the binding of a signal
molecule .

Regulatory proteins

Three domain (at least two)
1.
DNA binding domain
•
2.
Bind to DNA
protein-protein interaction domain
•
Interact with RNA polymerase, other regulatory
proteins, or other subunits of the same regulatory
protein.
dimerization domain
Domain—An independently folded part of a protein.
3.


Within regulatory proteins, the
amino acid side chains most
often hydrogen-bonding to
bases in the DNA are those of
Asn, Gln, Glu, Lys, and Arg
residues.
To interact with bases in the
major groove of DNA, a
protein requires a relatively
small structure that can stably
protrude from the protein
surface.
DNA-binding sites
The DNA-binding sites for regulatory
proteins are often inverted repeats of a
short DNA sequence (a palindrome) at
which multiple (usually two) subunits of a
regulatory protein bind cooperatively.
 The Lac repressor is unusual in that it
functions as a tetramer, with two dimers
tethered together at the end distant from the
DNA-binding sites.

Relationship between the lac operator
sequence and the lac promoter.
palindrome
AATTGT…ACAATT
TTAACA…TGTTAA

DNA binding domain :
DNA-binding sites :a short DNA
sequence (a palindrome)
 helix-turn-helix
 zinc finger
 homeodomain—found in some
eukaryotic proteins.

helix-turn-helix
This DNA-binding motif is crucial to the
interaction of many prokaryotic regulatory
proteins with DNA, and similar motifs occur in
some eukaryotic regulatory proteins.
 The helix-turn-helix motif comprises about 20
amino acids in two short a -helical segments,
each seven to nine amino acid residues long,
separated by a b turn

Helix-turn-helix
DNA-binding domain of the Lac repressor.
The helix-turn-helix motif is shown in red
and orange; the DNA recognition helix is red.

Zinc Finger
In a zinc finger, about 30 amino acid residues
form an elongated loop held together at the
base by a single Zn2+ ion, which is coordinated
to four of the residues (four Cys, or two Cys and
two His).
 The zinc does not itself interact with DNA;
rather, the coordination of zinc with the amino
acid residues stabilizes this small structural
motif. Several hydrophobic side chains in the
core of the structure also lend stability.

Zinc fingers. Three zinc fingers (gray) of the
regulatory protein Zif268, complexed with DNA
(blue and white) . Each Zn2+ (maroon) coordinates
with two His and two Cys residues (not shown).
 Homeodomain


Another type of DNA-binding domain has been
identified in a number of proteins that function as
transcriptional regulators, especially during eukaryotic
development.
This domain of 60 amino acids—called the
homeodomain, because it was discovered in homeotic
genes (genes that regulate the development of body
patterns)—is highly conserved and has now been
identified in proteins from a wide variety of organisms,
including humans . The DNA-binding segment of the
domain is related to the helix-turn-helix motif. The
DNA sequence that encodes this domain is known as the
homeobox.
Homeodomain.
Shown here is a homeodomain bound to DNA; one of
the helices (red), stacked on two others, can be seen
protruding into the major groove . This is only a small
part of the much larger protein Ultrabithorax (Ubx),
active in the regulation of development in fruit flies.

Motif—


An independent folding unit, or particular
structure, that recurs in many molecules. (DNA
or protein)
Domain—

An independently folded part of a protein.
 protein-protein
interaction domain:
Mediate interaction with RNA
polymerase, other regulatory
proteins, or other subunits of the
same regulatory protein.
– leucine zipper
– basic helix-loop-helix.
Leucine Zipper


This motif is an amphipathic helix with a series of
hydrophobic amino acid residues concentrated on
one side , with the hydrophobic surface forming
the area of contact between the two polypeptides
of a dimer.
A striking feature of these helices is the
occurrence of Leu residues at every seventh
position, forming a straight line along the
hydrophobic surface. Although researchers
initially thought the Leu residues interdigitated
(hence the name “zipper”)
Leucine zippers
(a) Comparison of amino acid sequences of
several leucine zipper proteins. Note the Leu (L)
residues at every seventh position in the zipper
region, and the number of Lys (K) and Arg (R)
residues in the DNA-binding region.
(b) Leucine zipper from the yeast activator
protein GCN4 (PDB ID 1YSA). Only the
“zippered” helices (gray and light blue),
derived from different subunits of the dimeric
protein, are shown. The two helices wrap
around each other in a gently coiled coil. The
interacting Leu residues are shown in red.
Basic Helix-Loop-Helix (bHLH)


Another common structural motif occurs in
some eukaryotic regulatory proteins implicated
in the control of gene expression during the
development of multicellular organisms.
These proteins share a conserved region of
about 50 amino acid residues important in both
DNA binding and protein dimerization. This
region can form two short amphipathic a helices
linked by a loop of variable length, the helixloop-helix.



distinct from the helix-turn-helix (motif
associated with DNA binding)
The helix-loop-helix motifs of two
polypeptides interact to form dimers.
In these proteins, DNA binding is mediated by
an adjacent short amino acid sequence rich in
basic residues, similar to the separate DNAbinding region in proteins containing leucine
zippers.
carboxyl-terminal end
Helix-loop-helix.
The human transcription factor Max,
bound to its DNA target site . The protein
is dimeric; one subunit is colored. The
DNA-binding segment (pink) merges with
the first helix of the helix-loop-helix (red).
The second helix merges with the
carboxyl-terminal end of the subunit
(purple). Interaction of the carboxylterminal helices of the two subunits
describes a coiled coil very similar to that
of a leucine zipper , but with only one pair
of interacting Leu residues (red side chains
near the top) in this particular example.
The overall structure is sometimes called a
helix-loop-helix/leucine zipper motif.
DNA-binding
SUMMARY
1. The expression of genes is regulated by
processes that affect the rates at which
gene products are synthesized and
degraded. Much of this regulation occurs
at the level of transcription initiation,
mediated by regulatory proteins that
either repress transcription (negative
regulation) or activate transcription
(positive regulation) at specific promoters.
2. Regulatory proteins are DNA-binding
proteins that recognize specific DNA
sequences; most have distinct DNAbinding domains. Within these
domains, common structural motifs
that bind DNA are the helix-turn-helix,
zinc finger, and homeodomain.
3. Regulatory proteins also contain domains
for protein-protein interactions, including
the leucine zipper and helix-loop-helix,
which are involved in dimerization, and
other motifs involved in activation of
transcription.
Regulation of gene expression
in prokaryotes
Structure of
Prokaryote

Genome is smaller than eukaryotes
No nucleus (DNA and a few
associated pro.) nucleoid


Gene cluster
Transcription and translation are
coupled

Polycistrons
Prokaryotes Provide Models for the Study of
Gene Expression in Mammalian Cells
 Regulation
at two levels
 Transcriptional
regulation
 Post-transcriptional regulation
 Operon
model
Two well-studied operons:
lac operon
trp operon
The concept of operon was introduced by Jacob
and Monod in 1961
Jacques Monod (1910–1976). Jacob
and Monod received the Nobel Prize in
Physiology or Medicine in 1965 for their
work on the genetic control of enzyme
synthesis.
François Jacob (1920–).
Many Prokaryotic Genes Are Clustered
and Regulated in Operons


Many prokaryotic mRNAs are polycistronic—
multiple genes on a single transcript—and the
single promoter that initiates transcription of the
cluster is the site of regulation for expression of all
the genes in the cluster.
The gene cluster and promoter, plus additional
sequences that function together in regulation, are
called an operon
Operon- is the coordinated unit of genetic expression in
bacteria. It is an operator plus two or more genes
under control of that operator. Occurs only in
prokaryotes (in eukaryotes, each gene is under separate
control).
Best known is the lac operon
The Structure of lactose operon

Promoter (P) RNA pol



Operator (O) Repressor



control sequence
site where the transcription enzyme initiates
transcription
Is a DNA sequence between the promoter and the
enzyme genes
Acts as an on and off switch for the genes
Structural Genes


One to several genes coding for enzymes of a
metabolic pathway
Translated simultaneously as a block
P
O
Representative prokaryotic operon
 Genes A, B, and C are transcribed on one
polycistronic mRNA.
 Typical regulatory sequences include binding sites
for proteins that either activate or repress
transcription from the promoter.
Operons—the basic concept of
Prokaryotic Gene Regulation
Regulated genes can be switched on and off
depending on the cell’s metabolic needs.
 Operon-a regulated cluster of adjacent
structural genes, operator site, promotor site,
and regulatory gene(s).

The lac Operon Is Subject to Negative
Regulation



The study of gene regulation began with the
lactose operon in E.coli. The operon model was
proposed to explain the regulation of RNA
synthesis related to lactose metabolism in E.coli
First introduced the concept of operon, operator,
repressor, inducer in gene regulation.
Jacob and Monod in 1961 described their operon
model in a classic paper.
The structure of Lac Operon


structural gene
 β-galactosidase (lacZ),
 galactoside permease(lacY)
 thiogalactoside transacetylase (lacA).
regulatory gene
 lac promoter P
 lac operator O
lacZ, lacY, lacA
P-Promoter, O-Operator I-Inhibitor
The roles of the three
structural genes

lacZ codes for the enzyme β-galactosidase, whose
active form is a tetramer of ~500 kD. The enzyme
breaks a β-galactoside into its component sugars.
 lacY codes for the β-galactoside permease, a 30
kD membrane-bound protein constituent of the
transport system. This transports β -galactosides
into the cell.
 lacA codes for β-galactoside transacetylase, an
enzyme that transfers an acetyl group from
acetyl-CoA to β-galactosides.
Lactose metabolism in E.
coli.
Uptake and metabolism of lactose
require the activities of
galactoside permease and βgalactosidase. Conversion of
lactose to allolactose by
transglycosylation is a minor
reaction also catalyzed by βgalactosidas

Each of these linked genes is transcribed into one
large mRNA molecule that contains multiple
independent translation start (AUG) and stop
(UAA) codons for each cistron. Thus, each protein
is translated separately, and they are not
processed from a single large precursor protein.
This type of mRNA molecule is called a
polycistronic mRNA.
How to write a gene and a protein?
 A gene is generally italicized in lower case
and the encoded protein, when abbreviated,
is expressed in roman type with the first
letter capitalized.
– For example, the gene lacI encodes the
repressor protein LacI.
Off
On

Several -galactosides structurally related to
allolactose are inducers of the lac operon but
are not substrates for -galactosidase; others
are substrates but not inducers. One
particularly effective and nonmetabolizable
inducer of the lac operon that is often used
experimentally is isopropylthiogalactoside
(IPTG):
The lac Operon Undergoes Positive
Regulation
The operator-repressor-inducer interactions
described earlier for the lac operon provide
an intuitively satisfying model for an on/off
switch in the regulation of gene expression.
 Operon regulation is rarely so simple
 Glucose affect the expression of the lac
genes

Glucose, metabolized directly by glycolysis,
is E. coli’s preferred energy source. Other
sugars can serve as the main or sole nutrient,
but extra steps are required to prepare them
for entry into glycolysis, necessitating the
synthesis of additional enzymes.
 Clearly, expressing the genes for proteins
that metabolize sugars such as lactose or
arabinose is wasteful when glucose is
abundant.

What happens to the expression of the lac
operon when both glucose and lactose are
present?


A regulatory mechanism known as catabolite
repression restricts expression of the genes
required for catabolism of lactose, arabinose, and
other sugars in the presence of glucose, even when
these secondary sugars are also present.
The effect of glucose is mediated by cAMP, as a
coactivator, and an activator protein known as
cAMP receptor protein, or CRP (the protein is
sometimes called CAP, for catabolite gene
activator protein).


CRP is a homodimer with binding sites for DNA
and cAMP. Binding is mediated by a helix-turnhelix motif within the protein’s DNA-binding
domain .
When glucose is absent, CRP-cAMP binds near
the lac promoter and stimulates RNA to a site
transcription 50-fold. CRP-cAMP is therefore a
positive regulatory element responsive to glucose
levels, whereas the Lac repressor is a negative
regulatory element responsive to lactose.
Activation of transcription of the lac operon by CRP
Sequence of the lac promoter compared with the promoter
consensus sequence. The differences mean that RNA polymerase
binds relatively weakly to the lac promoter until the polymerase
is activated by CRP-cAMP.
Regulation of
transcription from
the lac operon of
E.coli.
Levels of Control of Lac Operon
Expression
3 Scenarios:
1. No Lactose around
–
Operon switched off, no mRNA regardless of [glucose]
2. Lactose present; glucose also present
–
–
–
The presence of lactose inactivates the repressor
 Transcription occurs
Glucose present  cAMP is low  CRP does not ‘help’
transcription
3. Lactose present; no glucose
–
–
–
–
The presence of lactose inactivates the repressor
 Transcription occurs
NO Glucose  cAMP is high  cAMP binds CRP
(becomes activated)  CRP binds & ‘Helps’
Transcription
High Level of transcription
Many Genes for Amino Acid Biosynthetic
Enzymes Are Regulated by Transcription
Attenuation
The genes for the enzymes needed to
synthesize a given amino acid are
generally clustered in an operon and are
expressed whenever existing supplies of
that amino acid are inadequate for
cellular requirements.
 When the amino acid is abundant, the
biosynthetic enzymes are not needed
and the operon is repressed.

trp operon


The E. coli tryptophan (trp) operon includes five
genes for the enzymes required to convert
chorismate to tryptophan. Note that two of the
enzymes catalyze more than one step in the
pathway.
The mRNA from the trp operon has a half-life of
only about 3 min, allowing the cell to respond
rapidly to changing needs for this amino acid. The
Trp repressor is a homodimer, each subunit
containing 107 amino acid residues .
The trp operon


When tryptophan is abundant it binds to the Trp
repressor, causing a conformational change that
permits the repressor to bind to the trp operator
and inhibit expression of the trp operon. The trp
operator site overlaps the promoter, so binding of
the repressor blocks binding of RNA polymerase.
This simple on/off circuit mediated by a repressor
is not the entire regulatory story.
Transcription attenuation mechanism


Different cellular concentrations of tryptophan can
vary the rate of synthesis of the biosynthetic
enzymes over a 700-fold range.
Once repression is lifted and transcription begins,
the rate of transcription is fine-tuned by a second
regulatory process, called transcription
attenuation, in which transcription is initiated
normally but is abruptly halted before the operon
genes are transcribed. The frequency with which
transcription is attenuated is regulated by the
availability of tryptophan and relies on the very
close coupling of transcription and translation in
bacteria.

The trp operon attenuation mechanism uses
signals encoded in four sequences within a
162 nucleotide leader region at the 5 end of
the mRNA, preceding the initiation codon of
the first gene. Within the leader lies a region
known as the attenuator, made up of
sequences 3 and 4.
Transcriptional attenuation in the trp operon.
Transcription is initiated at the beginning of
the 162 nucleotide mRNA leader encoded by a
DNA region called trpL . A regulatory
mechanism determines whether transcription
is attenuated at the end of the leader or
continues into the structural genes.
Regulation of gene expression
in eukaryotes

Genome is bigger
Nucleus (DNA and
histone , nucleosome ,
chromatin)

Transcription and
translation is separated.

Structure of
Eukaryote
Post-transcriptional
modification

Split gene (Exon and
Intron)



Each cell of the higher organisms contains
the entire genome.
Gene expression in eukaryotes is regulated
to provide the appropriate response to
biological needs.
1.
2.
3.
Expression of certain genes (housekeeping
gene) in most of cells.
Activation of selected genes upon demand.
Permanent inactivation of several genes in all
but a few types.
In case of prokaryotic cells, most of the
DNA is organized into genes which can be
transcribed.
 In contrast, in mammals, very little of the
total DNA is organized into genes and their
associated regulatory sequence.
 Eukaryotic gene expression and its
regulation are highly complex!!!

Four important features of the regulation
of gene expression in eukaryotes


First, access to eukaryotic promoters is restricted
by the structure of chromatin, and activation of
transcription is associated with many changes in
chromatin structure in the transcribed region.
Second, although eukaryotic cells have both
positive and negative regulatory mechanisms,
positive mechanisms predominate in all systems
characterized so far. Thus, given that the
transcriptional ground state is restrictive,
virtually every eukaryotic gene requires
activation to be transcribed.
Third, eukaryotic cells have larger, more
complex multimeric regulatory proteins
than do bacteria.
 Finally, transcription in the eukaryotic
nucleus is separated from translation in the
cytoplasm in both space and time.

The Greater Complexity of Eukaryotic
Genomes Requires Elaborate
Mechanisms for Gene Regulation
 Gene
regulation is significantly more
complex in eukaryotes than in
prokaryotes for a number of reasons.

First, the genome being regulated is significantly
larger.
– The E. coli genome consists of a single, circular
chromosome containing 4.6 Mb. This genome encodes
approximately 2000 proteins.
– In comparison, one of the simplest eukaryotes,
Saccharomyces cerevisiae (baker's yeast), contains 16
chromosomes ranging in size from 0.2 to 2.2 Mb . The
yeast genome totals 17 Mb and encodes approximately
6000 proteins.
– The genome within a human cell contains 23 pairs of
chromosomes ranging in size from 50 to 250 Mb.
Approximately 40,000 genes are present within the
3000 Mb of human DNA.


It would be very difficult for a DNA-binding
protein to recognize a unique site in this vast
array of DNA sequences. Consequently, moreelaborate mechanisms are required to achieve
specificity.
Another source of complexity in eukaryotic
gene regulation is the many different cell types
present in most eukaryotes. (differentiation)
– Liver and pancreatic cells, for example, differ
dramatically in the genes that are highly expressed.
Moreover, eukaryotic genes are not
generally organized into operons. Instead,
genes that encode proteins for steps within a
given pathway are often spread widely
across the genome.
 Finally, transcription and translation are
uncoupled in eukaryotes, eliminating some
potential gene-regulatory mechanisms.

Chromatin structure and gene
expression
The DNA in higher organisms is extensively
folded and packed to form protein-DNA
complex called chromatin.
 The structural organization of DNA in the
form of chromatin plays an important role
in eukaryotic gene expression.

Condense
Decondense
Transcriptionally Active Chromatin Is
Structurally Distinct from Inactive Chromatin
Several forms of chromatin can be found ：
 About 10% of the chromatin in a typical
eukaryotic cell is in a more condensed form than
the rest of the chromatin. This form,
heterochromatin, is transcriptionally inactive.
 Heterochromatin is generally associated with
particular chromosome structures—the
centromeres, for example.
 The remaining, less condensed chromatin is called
euchromatin.
 Transcription
of a eukaryotic gene is
strongly repressed when its DNA is
condensed within heterochromatin.
Some, but not all, of the euchromatin is
transcriptionally active.
Types of Chromatin
Heterochromatin
Euchromatin
highly condensed
less condensed
during interphase, during interphase,
not actively
able to be
transcribed
transcribed
Nucleosomes Are Complexes of DNA and
Histones

The DNA in eukaryotic chromosomes is not
bare. Rather eukaryotic DNA is tightly bound
to a group of small basic proteins called
histones.

Five major histones are present in chromatin:




H2A, H2B, H3, and H4 (histone octamer)
H1
Histones have strikingly basic properties because a
quarter of the residues in each histone is either
arginine or lysine
The entire complex of a cell's DNA and
associated protein is called chromatin.


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
octamer. These repeating units are known as
nucleosomes.
This smaller complex of the histone octamer
and the 145-bp DNA fragment is the
nucleosome core particle. The DNA connecting
core particles in undigested chromatin is called
linker DNA. Histone H1 binds, in part, to the
linker nDNA.
Levels of Chromatin Structure
Eukaryotic DNA Is Wrapped Around
Histones to Form Nucleosomes

The eight histones in the core are
arranged into a (H3)2(H4)2 tetramer and a
pair of H2A/H2B dimers. The tetramer
and dimers come together to form a lefthanded superhelical ramp around which
the DNA wraps.

Each histone has an amino-terminal tail
that extends out from the core structure.
These tails are flexible and contain a
number of lysine and arginine residues. As
we shall see, covalent modifications of these
tails play an essential role in modulating the
affinity of the histones for DNA and other
properties.
Histone modifications
•Phosphorylaiton
•Acetylation
•Methylation
•Ubiqitination
•SUMOlation
•ADP-ribosylation
The acetylation and deacetylation of
histones figure prominently in the processes
that activate chromatin for transcription. As
noted above, the amino-terminal domains of
the core histones are generally rich in Lys
residues. Particular Lys residues are
acetylated by histone acetyltransferases
(HATs).
 Where chromatin is being activated for
transcription, the nucleosomal histones are
further acetylated by nuclear HATs.

Enhancers Can Stimulate Transcription by
Perturbing Chromatin Structure


Enhancer, DNA sequences, although they
have no promoter activity of their own,
greatly increase the activities of many
promoters in eukaryotes, even when the
enhancers are located at a distance of several
thousand base pairs from the gene being
expressed.
Enhancers function by serving as binding sites
for specific regulatory proteins. An enhancer
is effective only in the specific cell types in
which appropriate regulatory proteins are
expressed.
In many cases, these DNA-binding proteins
influence transcription initiation by
perturbing the local chromatin structure to
expose a gene or its regulatory sites rather
than by direct interactions with RNA
polymerase.
 This mechanism accounts for the ability of
enhancers to act at a distance.

Many Eukaryotic Promoters Are
Positively Regulated
Positive regulation?—— The storage of DNA
within chromatin effectively renders most
promoters inaccessible, so genes are normally
silent in the absence of other regulation. The
structure of chromatin affects access to some
promoters more than others, but repressors
that bind to DNA so as to preclude access of
RNA polymerase (negative regulation) would
often be simply redundant.
DNA-Binding Transactivators and Coactivators
Facilitate Assembly of the General Transcription
Factors

Successful binding of active RNA polymerase II
holoenzyme at one of its promoters usually
requires the action of other proteins :
1. basal transcription factors , required at every Pol II
promoter;
2. DNA binding transactivators, which bind to enhancers
or UASs and facilitate transcription; and
3. coactivators. The latter group act indirectly—not by
binding to the DNA—and are required for essential
communication between the DNA-binding
transactivators and the complexcomposed of Pol II and
the general transcription factors.
Protein—protein interaction!
TBP--TATA-binding protein




Eukaryotic Gene Expression Can Be Regulated by
Intercellular and Intracellular Signals
Regulation Can Result from Phosphorylation of
Nuclear Transcription Factors
Many Eukaryotic mRNAs Are Subject to
Translational Repression
Development Is Controlled by Cascades of
Regulatory Proteins
SUMMARY
1. The expression of genes is regulated by processes
that affect the rates at which gene products are
synthesized and degraded. Much of this
regulation occurs at the level of transcription
initiation, mediated by regulatory proteins that
either repress transcription (negative regulation)
or activate transcription (positive regulation) at
specific promoters.
2. In bacteria, genes that encode products with
interdependent functions are often clustered in
an operon, a single transcriptional unit.
Transcription of the genes is generally blocked
by binding of a specific repressor protein at a
DNA site called an operator. Dissociation of the
repressor from the operator is mediated by a
specific small molecule, an inducer. These
principles were first elucidated in studies of the
lactose (lac) operon. The Lac repressor
dissociates from the lac operator when the
repressor binds to its inducer, allolactose.
3. Regulatory proteins are DNA-binding
proteins that recognize specific DNA
sequences; most have distinct DNAbinding domains. Within these
domains, common structural motifs
that bind DNA are the helix-turn-helix,
zinc finger, and homeodomain.
4. Regulatory proteins also contain domains
for protein-protein interactions, including
the leucine zipper and helix-loop-helix,
which are involved in dimerization, and
other motifs involved in activation of
transcription.
5. In eukaryotes, positive regulation is more
common than negative regulation, and
transcription is accompanied by large
changes in chromatin structure.
Promoters for Pol II typically have a
TATA box and Inr sequence, as well as
multiple binding sites for DNA-binding
transactivators.
Operon





Structural gene--gene that codes for a
polypeptide
Promoter region--controls access to the
structural genes, located between the
promoter and structural genes, contains the
operator site.
Operator Site--region where the repressor
attaches
Regulatory genes--codes for repressor proteins
Polycistronic mRNA--transcript for several
polypeptides
REVIEW QUESTIONS
For each question, choose the ONE BEST answer
The lac operon is transcribed when
A. lactose is present and glucose is absent.
B. cAMP concentrations in the cell are high.
C. The cAMP–CAP protein is bound to the lac promoter
region.
D. the lac repressor is bound to allolactose or a similar
shaped molecule.
E. all of the above.
How lac operon works?
– Negative regulation
– Positive regulation
Key Terms
1.
2.
3.
4.
5.
6.
7.
Gene
Gene expression
Housekeeping gene
Operon
Operator
Promoter
Enhancer