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Chapter 18
Regulation of Gene Expression
Overview: Conducting the Genetic Orchestra
• Prokaryotes and eukaryotes alter gene expression
in response to their changing environment
• In multicellular eukaryotes, gene expression
regulates development and is responsible for
differences in cell types
Eye
Leg
Antenna
Wild type
Mutant
Bacteria regulate their gene expression
• Feedback mechanisms allow control over metabolism
so that cells produce only the products needed at that
time (with some limitations)
• This metabolic control occurs on two levels:
1. adjusting activity of metabolic enzymes
2. regulating genes that encode metabolic enzymes
Regulation of a
Metabolic Pathway
Feedback
inhibition
Precursor
trpE gene
Enzyme 1
In the pathway for
tryptophan synthesis,
an abundance of
tryptophan can both
(a) inhibit the activity of
the first enzyme
in the pathway
(feedback inhibition),
a rapid response, and
(b) repress expression of
the genes for all the
enzymes needed for
the pathway, a
longer-term response.
trpD gene
Enzyme 2
Regulation
of gene
expression
trpC gene
trpB gene
Enzyme 3
trpA gene
Tryptophan
(a) Regulation of
enzyme activity
(b) Regulation of
enzyme production
•
In bacteria, genes are often clustered into operons, composed of:
1. an operator, an “on-off” switch
2. a promoter
3. genes for metabolic enzymes
•
An operon can be switched off by a protein called a repressor
–
binds only to the operator
trp operon
Promoter
Promoter
Genes of operon (5)
DNA
Regulatory
gene
mRNA
trpE
trpR
3
trpC
trpB
trpA
Operator
Start codon Stop codon
no tryptophan
RNA
polymerase
mRNA 5
repressor
inactive
5
E
Protein
trpD
Repressor
D
C
B
A
Polypeptides (5) that make up
enzymes for tryptophan synthesis
(inactive)
Tryptophan absent, repressor inactive, operon on
operon
ON
Operons
•
A repressible operonis one that is usually on;
binding of a repressor to the operator shuts off transcription
– the trp operon is a repressible operon
– repressible enzymes usually function in anabolic pathways
• cells suspend production of an end product that is not needed
The trp operon
tryptophan
repressor
active
operon
OFF
DNA
No RNA made
mRNA
Active
repressor
Protein
Tryptophan
(corepressor)
corepressora small molecule that cooperates with a
repressor to switch an operon off
Tryptophan present, repressor active, operon off
Operons
•
A repressible operonis one that is usually on;
binding of a repressor to the operator shuts off transcription
– the trp operon is a repressible operon
– repressible enzymes usually function in anabolic pathways
• cells suspend production of an end product that is not needed
•
An inducible operonis one that is usually off;
a molecule, an inducer, inactivates the repressor & turns on transcription
– the classic example of an inducible operon is the lac operon
– inducible enzymes usually function in catabolic pathways
• cells only produce enzymes when there’s a nutrient that needs to be broken
down
The lac operon
Promoter
Regulatory
gene
Operator
lacl
DNA
lacZ
No
RNA
made
no lactose
3
mRNA
5
RNA
polymerase
repressor
active
operon
OFF
Protein
Active
repressor
Lactose absent, repressor active, operon off
The lac repressor is innately active, and in the absence of lactose
it switches off the operon by binding to the operator.
Allolactose, an isomer of lactose, derepresses the operon by inactivating the repressor.
In this way, the enzymes for lactose utilization are induced.
lac operon
3 genes
DNA
lacZ
lacl
3
mRNA
5
lacA
Permease
Transacetylase
RNA
polymerase
mRNA 5
-Galactosidase
Protein
Allolactose
(inducer)
lacY
Inactive
repressor
E.Coli uses 3 enzymes to take up and metabolize lactose
β-galactosidase: hydrolyzes lactose to glucose and galactose
Lactose present, repressor inactive, operon on permease: transports lactose into the cell
transacetylase: function in lactose metabolism is still unclear
inducera small molecule that
inactivates the repressor
lactose
repressor
inactive
operon
ON
Gene Regulation
• Regulation of the trp and lac operons involves
negative control of genes because operons are
switched off by the active form of the repressor
• Some operons are also subject to positive control
through a stimulatory activator protein,
such as catabolite activator protein (CAP)
• The lac operon is under dual control:
– negative control by the lac repressor
– positive control by CAP
Positive Control
•
When glucose (a preferred food source of E. coli ) is scarce,
the lac operon is activated by the binding of CAP
Promoter
DNA
glucose
lacl
lacZ
CAP-binding site
cAMP
Active
CAP
cAMP
Inactive
CAP
Lactose present, glucose scarce (cAMP level high):
abundant lac mRNA synthesized
RNA
Operator
polymerase
can bind
and transcribe
Inactive lac
repressor
When glucose levels increase, CAP detaches from the lac operon, turning it off
Promoter
DNA
lacl
lacZ
CAP-binding site
Operator
RNA
polymerase
can’t bind
Inactive
CAP
Lactose present, glucose present (cAMP level low):
little lac mRNA synthesized
Inactive lac
repressor
REVIEW
Repressible Operon
Genes not expressed
Genes expressed
Promoter
Genes
Operator
Active repressor:
corepressor bound
Inactive repressor:
no corepressor present
Corepressor
Inducible Operon
Genes not expressed
Genes expressed
Promoter
Operator
Genes
Active repressor:
no inducer present
Inactive repressor:
inducer bound
Eukaryotic Genomes
• Two features of eukaryotic genomes are
a major information-processing challenge:
1. the typical eukaryotic genome is much larger than that
of a prokaryotic cell
2. cell specialization limits the expression of many genes
to specific cells
The DNA-protein complex, called chromatin,
is ordered into higher structural levels than
the DNA-protein complex in prokaryotes
Differential Gene Expression
• Almost all the cells in an organism are genetically identical
• Differences between cell types result from
differential gene expression,
the expression of different genes by cells with the same genome
• Errors in gene expression can lead to diseases including cancer
Signal
Gene Expression
•
Gene expression is
regulated at many stages
NUCLEUS
Chromatin
DNA
Chromatin modification:
DNA unpacking involving
histone acetylation and
DNA demethylation
#1
Gene available
for transcription
Gene
Transcription
RNA
Exon
Primary transcript
#2 ***** most important
control point
Intron
#3
RNA processing
Cap
Tail
mRNA in nucleus
Transport to cytoplasm
CYTOPLASM
#4
Degradation
of mRNA
mRNA in cytoplasm
Translation
#5
Polypeptide
Protein processing, such
as cleavage and
chemical modification
#6
Degradation
of protein
Active protein
Transport to cellular
destination
Cellular function (such
as enzymatic activity,
structural support)
#6
#1
•
•
•
Genes within highly packed
heterochromatin
(highly condensed areas)
are usually not expressed
Chemical modifications to
histones and DNA of chromatin
influence both
chromatin structure and
gene expression
histone acetylationacetyl groups (-COCH3) are
attached to positively charged
lysines in histone tails
•
This process seems to
loosen chromatin structure,
thereby promoting the
initiation of transcription
Histones
#1
#2
#3
#4
#5
#6
Histone
tails
DNA
double helix
Amino acids
available
for chemical
modification
Histone tails protrude outward from a nucleosome
Unacetylated histones
Acetylated histones
Acetylation of histone tails promotes loose chromatin
structure that permits transcription
DNA Methylation
#1
• DNA methylationthe addition of methyl groups to certain bases in DNA,
is associated with reduced transcription in some species
• DNA methylation can cause long-term inactivation of genes
in cellular differentiation
– In genomic imprinting, methylation turns off either the maternal
or paternal alleles of certain genes at the start of development
Summary- Chromatin Modifications
• Although the chromatin modifications just discussed 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
• 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
#1
Eukaryotic Gene
•
#2 & #3
Associated with most eukaryotic genes are multiple control elements,
segments of noncoding DNA that help regulate transcription by binding
certain proteins
– Control elements & the proteins they bind are critical to the precise
regulation of gene expression in different cell types
Enhancer
(distal control elements)
Proximal
control elements
Exon
Intron
Exon
Poly-A signal Termination
sequence
region
Intron Exon
DNA
Upstream
Downstream
Promoter
Primary RNA
5
transcript
(pre-mRNA)
#1
Transcription
Exon
Intron
#2
Intron RNA
#3
#4
#5
#6
Poly-A signal
Exon
Intron Exon
Cleaved 3 end
of primary
transcript
RNA processing:
Cap and tail added;
introns excised and
exons spliced together
Coding segment
mRNA
3
5 Cap
5 UTR
(untranslated
region)
Start
codon
Stop
codon
Poly-A
3 UTR
(untranslated tail
region)
To initiate transcription, eukaryotic RNA polymerase requires the
assistance of proteins called transcription factors (TFs)
– General TFs are essential for the transcription of all protein-coding genes
– In eukaryotes, high levels of transcription of particular genes depend on
control elements interacting with specific TFs
 proximal control elementsare located close to the promoter
 distal control elementsgroups of which are called enhancers,
may be far away from a gene
or even in an intron
activatorspecific TF,
a protein that binds
to an enhancer & stimulates
transcription of a gene
repressorspecific TF,
inhibit expression of a gene
Activator proteins bind
to distal control elements
grouped as an enhancer in
the DNA. This enhancer has
three binding sites.
A DNA-bending protein brings
the bound activators closer to
the promoter. Other transcription
factors, mediator proteins, and
RNA polymerase are nearby.
The activators bind to certain
general transcription factors and
mediator proteins, helping them
form an active transcription
initiation complex on the promoter.
#2
Combinatorial Control
of Gene Activation
Enhancer
Control
elements
A particular combination of
control elements can activate
transcription only when the
appropriate activator proteins
are present
#2
Promoter
Albumin gene
Crystallin
gene
LENS CELL
NUCLEUS
LIVER CELL
NUCLEUS
Available
activators
Available
activators
Albumin gene
not expressed
Albumin gene
expressed
Crystallin gene
not expressed
(a) Liver cell
Crystallin gene
expressed
(b) Lens cell
Coordinately Controlled Genes
• Unlike the genes of a prokaryotic operon,
each of the co-expressed eukaryotic genes has
a promoter and control elements
• These genes can be scattered over
different chromosomes, but each has the
same combination of control elements
• Copies of the activators recognize
specific control elements and promote
simultaneous transcription of the genes
#2
#3-6
Mechanisms of Post-Transcriptional Regulation
• Transcription alone does not account for gene expression
• More and more examples are being found of
regulatory mechanisms that operate at various stages
after transcription
• Such mechanisms allow a cell to fine-tune gene expression
rapidly in response to environmental changes
Alternative RNA Splicing
Exons
DNA
Troponin T gene
Primary
RNA transcript
different mRNA molecules are produced
from the same primary transcript,
depending on which RNA segments
are treated as exons/introns
mRNA
RNA splicing
or
#3
mRNA Degradation
#4
• The life span of mRNA molecules in the cytoplasm
is a key to determining protein synthesis
• Eukaryotic mRNA is more long lived than prokaryotic mRNA
• Nucleotide sequences that influence the lifespan of mRNA
in eukaryotes reside in the untranslated region (UTR)
at the 3’ end of the molecule
Initiation of Translation
• The initiation of translation of selected mRNAs
– can be blocked by regulatory proteins that bind to
specific sequences or structures of the mRNA
• Alternatively, translation of all mRNAs
in a cell may be regulated simultaneously
– For example, translation initiation factors are
simultaneously activated in an egg following fertilization
#5
Protein Processing and Degradation
•
#6
After translation, various types of protein processing,
including cleavage and the addition of chemical groups, are subject to control
#1
#2
#3
#4
Multiple ubiquitin
molecules are attached
to a protein by enzymes
in the cytosol.
proteasomesare giant protein complexes that bind
protein molecules and degrade them
#5
#6
The ubiquitin-tagged protein
is recognized by a proteasome,
which unfolds the protein and
sequesters it within a central cavity.
Proteasome
and ubiquitin
to be recycled
Ubiquitin
Proteasome
Protein to
be degraded
Ubiquitinated
protein
Protein entering a
proteasome
Enzymatic
components of
the proteasome
cut the protein
into small
peptides, which
can be further
degraded by
other enzymes in
the cytosol.
Protein
fragments
(peptides)
REVIEW
Transcription
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.
• 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.
• Coordinate regulation:
Enhancer for
Enhancer for
liver-specific genes
lens-specific genes
Chromatin modification
Transcription
RNA processing
RNA processing
• Alternative RNA splicing:
Primary RNA
transcript
mRNA
degradation
Translation
Protein processing
and degradation
mRNA
or
Translation
• Initiation of translation can be controlled
via regulation of initiation factors.
mRNA degradation
• Each mRNA has a
characteristic life span,
determined in part by
sequences in the 5 and
3 UTRs.
Protein processing and degradation
• Protein processing and
degradation by proteasomes
are subject to regulation.
Noncoding RNAs play multiple roles
in controlling gene expression
• Only a small fraction of DNA codes for proteins,
and a very small fraction of the non-protein-coding DNA
consists of genes for RNA such as rRNA and tRNA
• A significant amount of the genome may be transcribed
into noncoding RNAs (ncRNAs)
• Noncoding RNAs regulate gene expression at two points:
• mRNA translation
• chromatin configuration
Hairpin
Hydrogen
bond
miRNA
Dicer
5 3
(a) Primary miRNA transcript
miRNA
miRNAprotein
complex
• MicroRNAs (miRNAs)small single-stranded
RNA molecules that can
bind to mRNA
•
These can degrade mRNA
or block its translation
mRNA degraded Translation blocked
(b) Generation and function of miRNAs
• The phenomenon of inhibition of gene expression by
RNA molecules is called
RNA interference (RNAi)
• RNAi is caused by
small interfering RNAs (siRNAs)
• siRNAs and miRNAs are similar but form from different RNA precursors
• In some yeasts siRNAs play a role in heterochromatin formation
and can block large regions of the chromosome
• RNA-based mechanisms may also block transcription of single genes
REVIEW
Chromatin modification
Chromatin modification
• Small or large noncoding RNAs can
promote the formation of heterochromatin
in certain regions, blocking transcription.
Transcription
RNA processing
mRNA
degradation
Translation
• miRNA or siRNA can block the translation
of specific mRNAs.
Translation
Protein processing
and degradation
mRNA degradation
• miRNA or siRNA can target specific
mRNAs for destruction.