Download Chapter 18: Regulation of Gene Expression

Chapter 18:
Regulation of Gene Expression
1. Gene Regulation in Bacteria
2. Gene Regulation in Eukaryotes
3. Gene Regulation in Development
4. Gene Regulation & Cancer
Gene Regulation
Gene regulation refers to all aspects of
controlling the levels and/or activities of
specific gene products.
• the gene product is either a protein or an RNA
molecule
• regulation can occur at any stage of gene
expression which involves
• accessibility of the gene itself (chromatin structure)
• transcription & translation (if gene encodes protein)
• modification of the gene product
Transcription Factors
Transcription factors are proteins that either help
activate or inhibit transcription.
Many transcription factors bind to specific DNA
sequences in the regulatory regions of genes.
Activation
domain
DNA-binding
domain
DNA
• DNA-binding
transcription
factors have a
DNA-binding
domain and one
or more activation
domains that
mediate effects
on transcription
1. Gene Regulation in Bacteria
Chapter Reading – pp. 361-364
Bacterial Gene Regulation
Gene regulation in bacteria is generally
accomplished at the levels of transcription and
post-translational modification of protein activity.
Bacterial genes are commonly organized in
multi-gene structures called operons:
• multiple gene coding regions organized in
sequence under control of a single promoter
• genes in the operon are part of same metabolic
pathway
• operons are typically inducible or repressible
Regulation of Tryptophan Production
Precursor
Feedback
inhibition
trpE gene
Enzyme 1
trpD gene
Enzyme 2
Regulation
of gene
expression
trpC gene

trpB gene

Enzyme 3
trpA gene
Tryptophan
(a) Regulation of enzyme
activity
• enzymes
involved in
tryptophan
synthesis are
part of a single
operon
(b) Regulation of enzyme
production
• regulation
involves
transcription &
posttranslational
modification
(feedback
inhibition)
trp operon
Promoter
Promoter
Genes of operon
DNA
trpE
trpR
trpD
trpC
trpB
trpA
C
B
A
Operator
Regulatory
gene
3
RNA
polymerase
Start codon
Stop codon
mRNA 5
mRNA
5
E
Protein
Inactive
repressor
D
Polypeptide subunits that make up
enzymes for tryptophan synthesis
(a) Tryptophan absent, repressor inactive, operon on
The trp Operon
DNA
No RNA
made
mRNA
Protein
Active
repressor
Tryptophan
(corepressor)
(b) Tryptophan present, repressor active, operon off
• trp repressor is inactive
unless bound to
tryptophan
• low tryptophan = ON
• high tryptophan = OFF
repressible operon
Regulatory
gene
DNA
Promoter
The lac Operon
Operator
lacI
lacZ
No
RNA
made
3
mRNA
RNA
polymerase
5
• low allolactose = OFF
Active
repressor
Protein
• lac repressor is active
unless bound to
allolactose
(a) Lactose absent, repressor active, operon off
• high allolactose = ON
lac operon
DNA
lacI
lacZ
lacY
lacA
RNA polymerase
3
mRNA
5
mRNA 5
-Galactosidase
Protein
Allolactose
(inducer)
Inactive
repressor
(b) Lactose present, repressor inactive, operon on
Permease
Transacetylase
inducible operon
…more on the
lac Operon
Promoter
DNA
lacI
lacZ
CAP-binding site
When ON the lac operon
is on “low” by default
If glucose (preferred
sugar) is unavailable,
lac operon is “turned up”
due to CAP activation
• cAMP is produced if
glucose is low
• cAMP binds and
activates CAP
• active CAP binds CAP
site increasing Tx
cAMP
Operator
RNA
polymerase
Active binds and
transcribes
CAP
Inactive
CAP
Allolactose
Inactive lac
repressor
(a) Lactose present, glucose scarce (cAMP level high):
abundant lac mRNA synthesized
Promoter
DNA
lacI
CAP-binding site
lacZ
Operator
RNA
polymerase less
likely to bind
Inactive
CAP
Inactive lac
repressor
(b) Lactose present, glucose present (cAMP level low):
little lac mRNA synthesized
2. Gene Regulation in Eukaryotes
Chapter Reading – pp. 365-376
Overview of Eukaryotic
Gene Regulation
Eukaryotic genes generally have the following:
• a single coding region consisting of exons & introns
• a single promoter
• multiple proximal and distal control sequences
• distal control sequences can be 1000s of base pairs away
Eukaryotic gene regulation is dependent on
chromatin structure in addition all stages
between transcription initiation and the
production of a functional gene product.
Signal
NUCLEUS
Chromatin
Chromatin modification:
DNA unpacking involving
histone acetylation and
DNA demethylation
DNA
Gene available
for transcription
Gene
Stages of Gene Regulation
Chromatin structure*
• controls access to genes
Transcription
RNA
Exon
Primary transcript
Intron
RNA processing
Cap
Tail
mRNA in nucleus
Transport to cytoplasm
CYTOPLASM
mRNA in cytoplasm
Degradation
of mRNA
• key stage of gene regulation
RNA processing*
• splicing of the RNA transcript
Translation
Polypeptide
Protein processing, such
as cleavage and
chemical modification
Degradation
of protein
Transcription
RNA stability
Translation of mRNA
Active protein
Transport to cellular
destination
Cellular function (such
as enzymatic activity,
structural support)
Post-translation modifications
*relevant to eukaryotes only
Chromatin Structure
Chromatin structure is regulated through
modifications of either the DNA itself or the
histone proteins associated with the DNA:
DNA modifications
• addition of methyl (CH3) groups to cytosines
• results in more compact, less accessible chromatin
• responsible for X-inactivation, genomic imprinting
Histone modifications
• addition of acetyl groups (“opens” chromatin)
• addition of CH3 (“closed”) or PO4 (“open”) groups
Histones & Chromatin Structure
Histone
tails
Amino acids
available
for chemical
modification
DNA
double
helix
Nucleosome
(end view)
(a) Histone tails protrude outward from a nucleosome
Acetylated histones
Unacetylated histones
(b) Acetylation of histone tails promotes loose chromatin
structure that permits transcription
DNA is wrapped
around histone
cores in structures
called nucleosomes.
• tails of histone
proteins in
nucleosomes can
have acetyl, methyl or
phosphate groups
added to induce a
more “open” or
“closed” chromatin
structure
Proximal & Distal Regulation
Enhancer
(distal control
elements)
Proximal
control
elements
Transcription
start site
Exon
DNA
Upstream
Distal elements
interact with
promoter due to
bending of DNA.
Intron
Exon
Intron
Downstream
Poly-A
signal
Intron Exon
Exon
Cleaved
3 end of
primary
RNA processing
transcript
Promoter
Primary RNA
transcript
(pre-mRNA)
Poly-A
Transcription
signal
sequence termination
region
Intron Exon
Transcription
Exon
5
Intron RNA
Coding segment
mRNA
G
P
P
5 Cap
AAA AAA
P
5 UTR
Start
codon
Stop
codon
3 UTR
3
Poly-A
tail
• control elements bind specific transcription factors
• can be located near the promoter (proximal) or very
far from the promoter (distal)
Promoter
Activators
DNA
Enhancer
Distal control
element
Current Model
of Eukaryotic
Transcription
Initiation
Gene
TATA box
General
transcription
factors
DNAbending
protein
Group of mediator proteins
RNA
polymerase II
Involves specific
transcription factors
as well as general
transcription
factors and other
proteins involved
in all transcription
Initiation.
RNA
polymerase II
Transcription
initiation complex
RNA synthesis
Differential
Gene
Expression
Different genes
are expressed in
different cell
types due to:
Enhancer
Control
elements
Promoter
Albumin gene
Crystallin
gene
LENS CELL
NUCLEUS
LIVER CELL
NUCLEUS
Available
activators
Available
activators
Albumin gene
not expressed
• differences in
transcription
factors
Albumin gene
expressed
• differences in
chromatin
structure
Crystallin gene
not expressed
(a) Liver cell
Crystallin gene
expressed
(b) Lens cell
Regulatory roles of non-coding RNA
Spliceosomes
• contain snRNA molecules that direct the process of
splicing introns from primary RNA transcripts
MicroRNAs (miRNA)
• complex with specific proteins to facilitate
destruction of specific mRNA molecules that contain
sequences complementary to miRNA sequence
• target chromatin modification to the centromeres of
chromosomes resulting in highly condensed
heterochromatin in the centromeres
• protection from infection by RNA viruses
Alternative Splicing of RNA
Exons
DNA
1
3
2
4
5
Troponin T gene
Primary
RNA
transcript
3
2
1
5
4
RNA splicing
mRNA
1
2
3
5
or
1
2
4
5
Hairpin
Hydrogen
bond
miRNA
Dicer
5 3
(a) Primary miRNA transcript
miRNA
miRNAprotein
complex
miRNA
Production
mRNA degraded Translation blocked
(b) Generation and function of miRNAs
Protein Degradation
Proteasome
and ubiquitin
to be recycled
Ubiquitin
Proteasome
Protein to
be degraded
Ubiquitinated
protein
Protein entering
a proteasome
Protein
fragments
(peptides)
• proteins to be degraded in cells (e.g., cyclins) are
“tagged” with a small protein called ubiquitin
• ubiquitinated proteins are directed to
proteosomes which then degrade them
Transcription
Chromatin modification
• Genes in highly compacted
chromatin are generally not
transcribed.
• Regulation of transcription initiation:
DNA control elements in enhancers bind
specific transcription factors.
• Histone acetylation seems
to loosen chromatin structure,
enhancing transcription.
• DNA methylation generally
reduces transcription.
Bending of the DNA enables activators to
contact proteins at the promoter, initiating
transcription.
• Coordinate regulation:
Enhancer for
liver-specific genes
Enhancer for
lens-specific genes
Chromatin modification
Transcription
RNA processing
RNA processing
• Alternative RNA splicing:
Primary RNA
transcript
mRNA
degradation
Translation
mRNA
Protein processing
and degradation
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.
Summary
of
Eukaryotic
Gene
Regulation
3. Gene Regulation in
Development
Chapter Reading – pp. 376-382
Embryonic Development
“From fertilization to fully developed organism.”
Involves regulation of
maternal and embryonic
gene expression:
1 mm
(a) Fertilized eggs of a frog
• maternal genes involved in
packaging the egg during
oogenesis (egg production)
2 mm
(b) Newly hatched tadpole
Eye
Leg
Antenna
Wild type
Mutant
• embryonic genes control
development after
fertilization
Mutations in either
maternal or embryonic
genes can result in
developmental defects
Key Events in Animal Development
Oogenesis
• egg production in the ovary results in essential gene
regulatory factors (RNA, protein) being packaged very
specifically and unevenly in the developing egg
Fertilization
• triggers translation of maternal mRNA and rapid
series of mitotic nuclear divisions (cleavage)
Gastrulation & Induction
• cell rearrangement and cell-cell signaling resulting
in the differentiation of cells and formation of distinct
body structures
Early Development
(a) Cytoplasmic determinants in the egg
(b) Induction by nearby cells
Unfertilized egg
Sperm
Fertilization
Cell-cell
communication also
induces changes in
gene expression.
Nucleus
Molecules of two
different cytoplasmic
determinants
Early embryo
(32 cells)
NUCLEUS
Zygote
(fertilized egg)
Mitotic
cell division
Two-celled
embryo
Egg is packaged
unevenly with
regulatory
factors that are
then partitioned
into different
cells after
fertilization.
Signal
transduction
pathway
Signal
receptor
Signaling
molecule
(inducer)
Head Thorax
Abdomen
(a) Adult
Follicle cell
1 Egg
developing within
ovarian follicle
Nucleus
Egg
0.5 mm
Nurse cell
Dorsal
BODY
AXES
Anterior
Left
Right
Posterior
2 Unfertilized egg
Depleted
nurse cells
Fertilization
Ventral
Early Drosophila
Development
• maternal genes determine
body axes and early pattern
formation
• embryonic genes eventually
take over and determine
subsequent morphogenesis
Egg
shell
Laying of egg
3 Fertilized egg
Embryonic
development
4 Segmented
embryo
0.1 mm
Body
segments
Hatching
5 Larval stage
(b) Development from egg to larva
Bicoid Determines Anterior End
The mutant phenotype named “Bicoid” results in
larva with 2 posteriors and no anterior (NO head!).
• due to a mutation Head
in the maternal
Bicoid gene
• Bicoid mRNA is
deposited in the
anterior end of
all eggs during
oogenesis
• Bicoid activates
anterior gene
expression after
fertilization
Tail
T1 T2 T3
A8
A6
A1 A2 A3 A4 A5
Wild-type larva
A7
250 m
Tail
Tail
A8
A8
A7
A6
A7
Mutant larva (bicoid)
Localization of Bicoid Protein, mRNA
RESULTS
Bicoid is a
Anterior end
morphogen of
maternal origin
100 m
Fertilization,
translation of
bicoid mRNA
Bicoid mRNA in mature
unfertilized egg
Bicoid mRNA in mature
unfertilized egg
Bicoid mRNA is
expressed into
protein after
fertilization
This results
in a Bicoid
“morphogen
gradient”
Bicoid protein in
early embryo
Bicoid protein in
early embryo
Nucleus
Embryonic
precursor cell
Master regulatory
gene myoD
Other muscle-specific genes
DNA
Myoblast
(determined)
OFF
OFF
mRNA
OFF
MyoD protein
(transcription
factor)
mRNA
MyoD
Part of a muscle fiber
(fully differentiated cell)
Specification of
vertebrate muscle tissue
mRNA
Another
transcription
factor
mRNA
mRNA
Myosin, other
muscle proteins,
and cell cycle–
blocking proteins
4. Gene Regulation & Cancer
Chapter Reading – pp. 383-388
Oncogenes
Oncogenes are genes with a role in cell cycle
progression that have undergone a mutation that
contributes to cancer formation (normal version
is called a proto-oncogene).
• generally due to dominant “gain-of-function”
mutations
• mutations are of 3 general types:
1) translocation of the gene
2) amplification (duplication) of the gene
3) mutations in the coding or regulatory regions
of the gene
More on Oncogenes
Proto-oncogene
DNA
Translocation or
transposition: gene
moved to new locus,
under new controls
Gene amplification:
multiple copies of
the gene
New
promoter
Normal growthstimulating
protein in excess
Point mutation:
within a control
within
element
the gene
Oncogene
Normal growth-stimulating
protein in excess
Normal growthstimulating
protein in
excess
Oncogene
Hyperactive or
degradationresistant
protein
• mutations that result in excessive expression or
function can contribute to cancer
MUTATION
1 Growth
factor
Ras
3 G protein
GTP
Ras
P
P
P
2 Receptor
P
P
P
Hyperactive Ras protein
(product of oncogene)
issues signals on its
own.
GTP
4 Protein kinases
(phosphorylation
cascade)
Ras is a G protein that
is a proto-oncogene.
NUCLEUS
5 Transcription
factor (activator)
Gain-of-function Ras
mutations can trigger
“signal-independent”
activation of cell cycle.
(a) Cell cycle–stimulating pathway
DNA
Gene expression
Protein that
stimulates
the cell cycle
Tumor Suppressor Genes
Tumor Suppressor Genes encode gene products
that inhibit cell cycle progression.
Mutations in tumor suppressor genes are
typically recessive “loss-of-function” mutations.
• typically requires 2 mutant alleles (recessive)
• loss of functional gene product leads to defect
in:
• inhibiting cell cycle progression
• triggering apoptosis
• activating DNA repair
2 Protein kinases
MUTATION
3 Active
form
of p53
UV
light
1 DNA damage
in genome
DNA
Protein that
inhibits
the cell cycle
(b) Cell cycle–inhibiting pathway
Defective or missing
transcription factor,
such as
p53, cannot
activate
transcription.
Cancer Requires Multiple Mutations
The “multi-step” or “multi-hit” hypothesis.
EFFECTS OF MUTATIONS
Protein
overexpressed
Protein absent
Colon
Cell cycle
overstimulated
Increased cell
division
Cell cycle not
inhibited
(c) Effects of mutations
1 Loss
of tumorsuppressor
gene APC
(or other)
2 Activation
of ras
oncogene
Colon wall
Normal colon
epithelial cells
4 Loss
of tumorsuppressor
gene p53
Small benign
growth
(polyp)
3 Loss
of tumorsuppressor
gene DCC
Larger
benign growth
(adenoma)
5 Additional
mutations
Malignant
tumor
(carcinoma)
Key Terms for Chapter 18
• nucleosome, euchromatin, heterochromatin
• operon, repressor, operator, repressible, inducible
• control elements, distal, proximal, enhancer
• general vs specific transcription factors
• mediator proteins, DNA bending protein
Relevant
Chapter
Questions
• miRNA, alternative RNA splicing, Dicer, hairpin
• oogenesis, cytoplasmic determinants, induction
• morphogenesis, morphogen, morphogen gradient
• oncogene, proto-oncogene, tumor suppressor gene
• gain-of-function, loss-of-function mutations
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