Regulation of Gene Expression-Chapter 15
• Prokaryotes and eukaryotes alter gene
expression in response to their changing
environment
• In multicellular eukaryotes, gene expression
also regulates development and is responsible
for differences in cell types
• Protein/DNA, RNA/DNA, and RNA/RNA
interactions all play roles in regulating gene
expression in eukaryotes
Bacteria often respond to environmental change by
regulating transcription
• Natural selection has favored bacteria that
produce only the products needed by that cell
• A cell can regulate the production of enzymes
by feedback inhibition or by gene regulation
• Which of these forms of regulation saves more
energy? Which can respond to changes more
quickly?
LE 18-20
Regulation of enzyme
activity
Precursor
Regulation of enzyme
production
Feedback
inhibition
Enzyme 1
Gene 1
Enzyme 2
Gene 2
Regulation
of gene
expression
Enzyme 3
Gene 3
Enzyme 4
Gene 4
Enzyme 5
Tryptophan
Gene 5
The Operon Model
• Gene expression in bacteria is controlled
by the operon model
• Jacob and Monod (1961)
• Operon elements-repressor gene;
operator; structural gene(s)
• Inducible and repressible operons
Operons: The Basic Concept
• A group of functionally related genes can be
coordinately controlled by a single “on-off
switch”
• The regulatory “switch” is a segment of DNA
called an operator usually positioned within
the promoter
• An operon is the entire stretch of DNA that
includes the operator, the promoter, and the
genes that they control
Repressible and Inducible Operons: Two
Types of Negative Gene Regulation
• A repressible operon is one that is usually on;
binding of a repressor to the operator shuts
off transcription
• The trp operon is a repressible operon
• An inducible operon is one that is usually off;
a molecule called an inducer inactivates the
repressor and turns on transcription
• The lac operon is an inducible operon
• Inducible enzymes usually function in
catabolic pathways; their synthesis is induced
by a chemical signal
• Repressible enzymes usually function in
anabolic pathways; their synthesis is
repressed by high levels of the end product
• Regulation of the trp and lac operons
involves negative control of genes because
operons are switched off by the active form of
the repressor
The repressible operon (trp operon)
• Shuts down a biochemical pathway when
the end product of the pathway build up.
• Normal status-transcription occurs
because the operator is unblocked.
• End product of the pathway (co-repressor)
alters shape of the repressor protein so
that it binds to the operator blocking
transcription of the structural genes
LE 18-21a
trp operon
Promoter
Promoter
Genes of operon
DNA
Regulatory
gene
mRNA
trpE
trpR
3
trpC
trpB
trpA
C
B
A
Operator
Start codon Stop codon
RNA
polymerase
mRNA 5
5
E
Protein
trpD
Inactive
repressor
D
Polypeptides that make up
enzymes for tryptophan synthesis
Tryptophan absent, repressor inactive, operon on
LE 18-21b_1
DNA
mRNA
Active
repressor
Protein
Tryptophan
(corepressor)
Tryptophan present, repressor active, operon off
LE 18-21b_2
DNA
No RNA made
mRNA
Active
repressor
Protein
Tryptophan
(corepressor)
Tryptophan present, repressor active, operon off
• By default the trp operon is on and the genes
for tryptophan synthesis are transcribed
• When tryptophan is present, it binds to the trp
repressor protein, which then turns the
operon off
• The repressor is active only in the presence
of its corepressor tryptophan; thus the trp
operon is turned off (repressed) if tryptophan
levels are high
Inducible operons
• Activates breakdown of a substrate when
the substrate becomes available.
• Normal status-transcription does not
occurs because the operator is blocked by
the repressor protein.
• Substrate (inducer) alters shape of the
repressor protein so that does not bind to
the operator allowing transcription of the
structural genes to occur
• The lac operon is an inducible operon and
contains genes that code for enzymes used in
the hydrolysis and metabolism of lactose
• By itself, the lac repressor is active and
switches the lac operon off
• A molecule called an inducer inactivates the
repressor to turn the lac operon on
• For the lac operon, the inducer is allolactose,
formed from lactose that enters the cell
LE 18-22a
Promoter
Regulatory
gene
Operator
lacl
DNA
lacZ
No
RNA
made
3
mRNA
5
Protein
RNA
polymerase
Active
repressor
Lactose absent, repressor active, operon off
LE 18-22b
lac operon
DNA
lacZ
lacl
3
mRNA
5
lacA
Permease
Transacetylase
RNA
polymerase
mRNA 5
-Galactosidase
Protein
Allolactose
(inducer)
lacY
Inactive
repressor
Lactose present, repressor inactive, operon on
Positive Gene Regulation
• The lac operon can be turned on or off by
the presence/absence of the inducer
molecule (lactose)
• CAP and CAMP can also regulate the rate
of transcription of the lac operon (volume
control versus on/off control)
• If both glucose and lactose are present,
the bacteria will preferentially use glucose
LE 18-23a
Promoter
DNA
lacl
lacZ
CAP-binding site
Active
CAP
cAMP
Inactive
CAP
RNA
Operator
polymerase
can bind
and transcribe
Inactive lac
repressor
Lactose present, glucose scarce (cAMP level high): abundant lac
mRNA synthesized
LE 18-23b
Promoter
DNA
lacl
CAP-binding site
Inactive
CAP
lacZ
Operator
RNA
polymerase
can’t bind
Inactive lac
repressor
Lactose present, glucose present (cAMP level low): little lac
mRNA synthesized
Positive Gene Regulation
• E. coli will preferentially use glucose when it is
present in the environment
• When glucose is scarce, CAP (catabolite activator
protein) acts as an activator of transcription
• CAP is activated by binding with cyclic AMP
(cAMP)
• Activated CAP attaches to the promoter of the lac
operon and increases the affinity of RNA
polymerase, thus accelerating transcription
• When glucose levels increase, CAP detaches
from the lac operon, and transcription
proceeds at a very low rate, even if lactose is
present
• CAP helps regulate other operons that
encode enzymes used in catabolic pathways
Differential Gene Expression in Eukaryotes
• Almost all the cells in an multicellular organism are
genetically identical
• Differences between cell types result from differential
gene expression, the expression of different genes by
cells with the same genome
• In a given cell, usually about 20% of its genes are active at
a given time (rest turned off)
• Errors in gene expression can lead to diseases including
cancer
• Gene expression is regulated at many stages in
eukaryotes (in addition to regulation at the transcriptional
level which bacteria also do)
Regulation of chromatin
Structure:
• DNA is packed into an elaborate complex known
as chromatin
• The basic unit of chromtain is the nucleosome
• Histone proteins help maintain nucleosome
structure
• The structural organization of chromatin packs
DNA into a compact form and also helps
regulate gene expression in several ways
Fig. 16-21b
Chromatid
(700 nm)
30-nm fiber
Loops
Scaffold
300-nm fiber
Replicated
chromosome
(1,400 nm)
30-nm fiber
Looped domains
(300-nm fiber)
Metaphase
chromosome
Fig. 16-21a
Nucleosome
(10 nm in diameter)
DNA
double helix
(2 nm in diameter)
H1
Histones
DNA, the double helix
Histones
Histone tail
Nucleosomes, or “beads
on a string” (10-nm fiber)
Chromatin Packing in
Eukaryotes
• Loosely packed DNA (euchromatin) is
usually transcribed
• Tightly packed DNA (heterochromatin) is
not usually transcribed
Regulation of Chromatin Structure
• Chemical modifications to histones and DNA of
chromatin influence both chromatin structure
(tightly packed versus loosely packed) and
gene expression
• Chemical modification of histones include
acetylation and methylation
• Chemical modification of DNA include
methylation
Histone Modifications
• In histone acetylation, acetyl groups
(-COCH3)are attached to positively charged
lysines in histone tails
• This process loosens chromatin structure,
thereby promoting the initiation of transcription
Fig. 18-7
Histone
tails
DNA
double helix
Amino
acids
available
for chemical
modification
(a) Histone tails protrude outward from a
nucleosome
Unacetylated histones
Acetylated histones
(b) Acetylation of histone tails promotes loose
chromatin structure that permits transcription
DNA Methylation
• DNA methylation, the addition of methyl groups (CH3) to
certain bases in DNA, usually cytosine, is associated with
reduced transcription in some species
• DNA methylation can cause long-term inactivation of genes
in cellular differentiation
• Barr Bodies
• Once methylated, genes usually remain so through
successive cell divisions
• After replication, enzymes methylate the correct daughter
strand so that the methylation pattern is inherited
Epigenetic Inheritance
• Though chromatin modifications do not alter
DNA sequence, they may be passed to future
generations of cells
• The inheritance of traits transmitted by
mechanisms not directly involving the
nucleotide sequence is called epigenetic
inheritance
• Epigenetic modifications can be reversed,
unlike mutations in DNA sequence
Epigenetic Inheritance
• Might explain why one twin gets a genetic
disease (schizophrenia) and the other
does not.
• Alterations in normal DNA methylation
patterns are seen in some cancers-these
changes can cause inappropriate gene
expression in cancer cells.
Regulation of Transcription Initiation
• Chromatin-modifying enzymes provide initial
control of gene expression by making a region
of DNA either more or less able to bind the
transcription machinery
• Regulation of transcription by enhancers (and
their associated control elements) is another
important mechanism by which initiation of
transcription can be regulated.
Organization of a Typical Eukaryotic Gene
• Associated with most eukaryotic genes are
control elements, segments of noncoding
DNA that help regulate transcription by binding
certain proteins
• Control elements and the proteins they bind
are critical to the precise regulation of gene
expression in different cell types
Figure 15.8
Enhancer
(distal control
elements)
Proximal
control
elements
Transcription
start site
Exon
Poly-A signal
sequence
Intron Exon
Transcription
termination
region
Intron Exon
DNA
Upstream
Downstream
Promoter
Transcription
Primary RNA
5
transcript
(pre-mRNA)
Exon
Intron RNA
Intron Exon
Poly-A
signal
Intron Exon
RNA processing
Cleaved 3
end of
primary
transcript
Coding segment
mRNA
G
P
P
AAA AAA
P
5 Cap 5 UTR
Stop
Start
codon codon
3 UTR
3
Poly-A
tail
The Roles of Transcription Factors
• To initiate transcription, eukaryotic RNA
polymerase requires the assistance of proteins
called transcription factors. React with proximal
control elements of the gene (DNA)
• General transcription factors are essential for the
transcription of all protein-coding genes
• If a gene is going to be transcribed at anything but
a slow rate, specific transcription factor must also
be involved.
• In eukaryotes, high levels of transcription of
particular genes depend on control elements
interacting with specific transcription factors
Enhancers and Specific Transcription Factors
• An activator is a protein (a specific transcription
factor) that binds to a (distal) control element of
an enhancer and stimulates transcription of a
gene
• Activators have two domains, one that binds DNA
and a second that activates transcription
• Bound activators cause mediator proteins to
interact with proteins at the promoter
Figure 15.9
Activation
domain
DNA
DNA-binding
domain
• Bound activators are brought into contact
with a group of mediator proteins through
DNA bending
• The mediator proteins in turn interact with
proteins at the promoter
• These protein-protein interactions help to
assemble and position the initiation complex
on the promoter
Figure 15.10-3
Promoter
Activators
DNA
Enhancer
Distal control
element
Gene
TATA box
General transcription
factors
DNAbending
protein
Group of mediator proteins
RNA
polymerase II
RNA
polymerase II
Transcription
initiation complex
RNA synthesis
Why does a liver cell produce albumin and a
lens cell crystalline protein?
• All activators needed for high-level
expression of the albumin gene are
present in liver cells but not lens cells.
• All activators needed for high-level
expression of the crystalline gene are
present in lens cells but not liver cells.
• The presence of activators in cells may
occur at precise times during development
or in a particular cell type
Figure 15.11
Enhancer
Control
elements
Enhancer
(a) LIVER CELL NUCLEUS
Available
activators
Albumin gene
expressed
Crystallin gene
not expressed
Promoter Albumin gene
Promoter Crystallin gene
(b) LENS CELL NUCLEUS
Available
activators
Albumin gene
not expressed
Crystallin gene
expressed
Mechanisms of Post-Transcriptional
Regulation
• Transcription alone does not account for gene
expression
• Regulatory mechanisms can operate at
various stages after transcription
• Such mechanisms allow a cell to fine-tune
gene expression rapidly in response to
environmental changes
Figure 15.UN02
Chromatin modification
Transcription
RNA processing
mRNA
degradation
Translation
Protein
processing
and degradation
RNA Processing
• In alternative RNA splicing, different mRNA
molecules are produced from the same primary
transcript, depending on which RNA segments are
treated as exons and which as introns
• In Drosophila-one gene has enough exons that
alternative splicing can produce 19,000 different
membrane proteins. Each nerve cell has a different
membrane protein. Estimates are that 90% of human
genes are alternatively spliced.
Figure 15.12
Exons
DNA
2
1
3
5
4
Troponin T gene
Primary
RNA
transcript
2
1
3
4
5
RNA splicing
mRNA
1
2
3
5
or
1
2
4
5
Figure 15.UN03
Chromatin modification
Transcription
RNA processing
mRNA
degradation
Translation
Protein
processing
and degradation
mRNA Degradation
• The life span of mRNA molecules in the cytoplasm
is important in determining the pattern of protein
synthesis in a cell
• Eukaryotic mRNA generally survives longer than
prokaryotic mRNA
• Nucleotide sequences that influence the life span of
mRNA in eukaryotes reside in the untranslated
region (UTR) at the 3 end of the molecule
• Noncoding rna’s can cause m-rna to be degraded
or have it inhibited (more about that later)
Protein Processing and Degradation
• After translation, various types of protein
processing, including cleavage and chemical
modification, are subject to control (pro-insulin
insulin)
• The length of time each protein functions in a
cell is regulated by means of selective
degradation.To mark a particular protein for
destruction, the cell commonly attaches
molecules of ubiquitin to the protein, which
triggers its destruction
Figure 15.UN06
Chromatin modification
• Genes in highly compacted
chromatin are generally not
transcribed.
• Histone acetylation seems
to loosen chromatin
structure,
enhancing
transcription.
• DNA methylation generally
reduces transcription.
Transcription
• Regulation of transcription initiation:
DNA control elements in enhancers bind
specific transcription factors.
Bending of
the DNA
enables
activators to
contact proteins at
the promoter, initiating transcription.
• The genes in a coordinately controlled
group all share a combination of control
elements.
Chromatin modification
Transcription
RNA processing
RNA processing
• Alternative RNA splicing:
Primary RNA
transcript
mRNA
mRNA
degradation
or
Translation
Translation
Protein processing
and degradation
• Initiation of translation can be controlled
via regulation of initiation factors.
mRNA degradation
Protein processing and degradation
• Each mRNA has a
characteristic life span.
• Protein processing and degradation are
subject to regulation.
Noncoding RNAs play multiple roles in
controlling gene expression
• Protein coding DNA only accounts for about 1.5%
of the human genome
• Only a small fraction of DNA codes for rRNA, and
tRNA. What is the rest of DNA for?
• A significant amount of the genome may be
transcribed into noncoding (nc)RNAs (rock star
versus backups)
• Noncoding RNAs regulate gene expression at the
level of translation by degrading M-rna or blocking
its translation
Effects on mRNAs by MicroRNAs and
Small Interfering RNAs
• MicroRNAs (miRNAs) are small singlestranded RNA molecules that can bind to
complementary mRNA sequences
• These can degrade the mRNA or block its
translation
Fig. 18-13
Hairpin
miRNA
Hydrogen
bond
Dicer
miRNA
5 3
(a) Primary miRNA transcript
mRNA degraded
miRNAprotein
complex
Translation blocked
(b) Generation and function of miRNAs
• Another class of small RNAs are called small interfering
RNAs (siRNAs)
• siRNAs and miRNAs are similar but form from different RNA
precursors
• The phenomenon of inhibition of gene expression by siRNAs
is called RNA interference (RNAi)
• RNAi may represent the evolution of a genetic control
system from a defense mechanism against double stranded
RNA virus infection.
• Researchers uses RNAi to knock out genes and study their
function.
Researchers can monitor expression of specific
genes
• Cells of a given multicellular organism differ
from each other because they express
different genes from an identical genome
• The most straightforward way to discover
which genes are expressed by cells of
interest is to identify the mRNAs being made
Studying the Expression of Single Genes
• We can detect mRNA in a cell using nucleic acid
hybridization, the base pairing of a strand of
nucleic acid to its complementary sequence
• The complementary molecule in this case is a short
single-stranded DNA or RNA called a nucleic acid
probe
• Each probe is labeled with a fluorescent tag to allow
visualization
• The technique allows us to see the mRNA in place
(in situ) in the intact organism and is thus called in
situ hybridization
Figure 15.14
50 m
• Genome-wide expression studies can be carried out using
DNA microarray assays
• A microarray—also called a DNA chip—contains tiny
amounts of many single-stranded DNA fragments affixed to
the slide in a grid
• The experiment can identify subsets of genes that are being
expressed differently in one sample (breast cancer tissue for
example)compared to another (normal tissue).
Figure 15.17
Genes in red
wells expressed
in first tissue.
Genes in green
wells expressed
in second tissue.
Genes in yellow
wells expressed
in both tissues.
DNA microarray
Genes in black
wells not
expressed in
either tissue.
Notes for microarray diagram
• Each little dot represent a well in a plate.
• Each well has a gene from the two tissues
tested. This plate has 5,760 wells (about 25% of
human genes).
• The plate is incubated with probes from the two
tissues (one tissues probes are labeled green
and the other red).
• Colors for each well indicate whether the gene is
expressed in one tissue (red or green), both
tissues (yellow) or neither (black)