Download Activation of Transcription

Control of Gene Expression
Shaw-Jenq Tsai
Department of Physiology
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Thinking about Gene Regulation
Humans begin life from a single cell; all the
genetic information needed to create an adult
is in our genome.
Embryonic cells undergo differentiation to
produce specific cell types such as muscle,
nerve, and blood cells.
Different cell types are the consequence of
differential gene expression.
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A typical differentiated mammalian cell makes
about 100,000 proteins from approximately
35,000 genes.
Most of these are housekeeping proteins needed to
maintain all cell types.
Certain proteins can only be detected in specific
cell types.
How is gene expression regulated?
Regulation of gene expression is very complex
Presently – we have a superficial understanding
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Control of Gene Expression
Synthesis of a protein involves discrete
steps
Several levels at which control mechanisms
work
Transcriptional control
RNA processing control
Translation control
Protein activity control
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Transcriptional level control
Differential gene transcription – the
major mechanism of selective protein
synthesis
Governed by a large number of proteins
known as transcription factors
Two functional classes of transcription
factors
General transcription factors
Specific transcription factors
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Specific transcription factors
A single gene controlled by many regulatory
sites – bind different regulatory proteins
A single regulatory protein may become
attached to numerous sites on the genome
Cells respond to environmental stimuli by
synthesizing different transcription factors
Bind to different sites on DNA
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Specific Transcription Factors
PEPCK
A key enzyme of gluconeogenesis (conversion of
pyruvate to glucose)
Synthesized in liver in response to low glucose
Synthesis drops sharply after a meal
Level of synthesis of PEPCK controlled by
different transcription factors
e.g. receptors for hormones involved in
regulating carbohydrate metabolism
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Promoter Structure
Closest upstream sequence
TATA box – major element of the gene’s promoter
Region from TATA box to start of transcription
site is the core promoter
Site of assembly of preinitiation complex – RNA
polymerase II and general transcription factors
Two other promoter sequences
CAAT box
GC box
Core
promoter
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Control of PEPCK Gene Expression
PEPCK
TATA box determines the site of initiation of
transcription
CAAT and GC boxes regulate the frequency of
transcription
All located within 100 to 150 base pairs upstream
of the transcription start site – proximal promoter
elements
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Activation of Transcription
Hormones which affect transcription of
PEPCK include insulin, thyroid hormone,
glucagon and glucocorticoids
All affect transcription factors which bind DNA
DNA sites bound by transcription factors are
termed – response elements
Glucocorticoids stimulate PEPCK expression
by binding to a specific DNA sequence
termed – a glucocorticoid response element
(GRE)
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Activation of Transcription
Same GRE is located upstream from
different genes on different chromosomes
Thus – a single stimulus – elevated
glucocorticoid concentrations –
simultaneously activates a range of genes
needed in a comprehensive response to
stress
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Enhancers
Activation of Transcription
Expression of genes also regulated by more distant DNA
elements termed enhancers
Can be experimentally moved without affecting their
ability to enhance gene expression
May be 1000s or 10000s base pairs upstream or
downstream from the gene
How??
Brought into close proximity to the gene as DNA can form loops
Promoters and enhancers cordoned off from other genes by
sequences called insulators
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Control of Gene Expression
Activation of Transcription
Transcription factor
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Control of Gene Expression
Activation of Transcription
Enhancers
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Action of an Insulator
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Figure 12.34
Two hypotheses for the mechanism of insulator
activity.
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Action of Transcription Factor
A transcription factor bound to an enhancer
may act via the following mechanisms:
Recruit general transcription factors and DNA
polymerase II to the core promoter
Stabilize the transcription machinery located in
the core promoter
Via an intermediary termed a coactivator
Coactivators are large complexes with 15 to 20 subunits
Do not directly bind DNA
Interact with a range of transcription factors
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Structure of Transcription Factors
Contain different domains which mediate the
different functions – at least two domains
DNA-binding domain
Activation domain
Commonly form dimers
Example
Glucocorticoid receptor
Binds DNA at the glucocorticoid response element (GRE)
Ligand-binding domain / DNA-binding domain /
Activation domain
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Transcription Factors Binding
Element
GRE
5’-AGAACAnnnTGTTCT-3’
3’-TCTTGTnnnACAAGA-5’
A palindrome
Two-fold nature is important
Pairs of GR polypeptides bind to DNA forming
dimers
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Transcription Factor Motifs
Transcription factors belong to each of several
classes based upon specific types of binding
domains or motifs
Many contain an a-helix which is inserted into
the DNA major groove
Recognizes the particular nucleotide sequence
lining the groove
Binding between aa and DNA (including DNA
backbone) via:
Van der Waals (hydrophobic) forces
Ionic bonds
And hydrogen bonds
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Control of Gene Expression
Transcription Factor Motifs
Common transcription factor motifs
Zinc finger
Helix-loop-helix
Leucine zipper
HMG box
Shared feature
Structurally stable framework
Specific DNA recognizing sequences are correctly positioned
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Types of DNA binding proteins
DNA and RNA polymerase
repair enzymes
structural proteins
transcription factors
DNA binding motifs
zinc fingers
leucine zippers
helix-turn-helix
helix-loop-helix
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Zinc finger
Zn ion coordinated to two cysteines and two
histidines
Each contains multiple zinc finger domains
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Helix-loop-helix (HLH)
Two a helices separated
by a loop
Often preceded by a
stretch of basic aa which
interact with a specific
nucleotide string
Always occur as dimers
homodimers
heterodimers
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Leucine zipper motif
Leucines every seven aa
along an a-helix
All leucines face the
same direction
Two a-helices can zip
together forming a
coiled coil
Basic aa on opposite
side of coils
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Repression of Transcription
Cells also possess negative regulatory
elements
Mechanisms:
Binding to promoter elements
Blocking assembly of the preinitiation complex
Inhibiting binding or functioning of transcriptional
activators
Modifying DNA and its interaction with nucleosomes
Some transcription factors activate some genes
and repress others
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Mechanisms of Transcription
Repression
Binding to promoter elements
Blocking assembly of the preinitiation complex
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Mechanisms of Transcription
Repression
Inhibiting binding or functioning of
transcriptional activators
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Repression of Transcription
DNA Methylation
Methyl groups may be attached
to cytosine (C5 position)
Methyltransferases
Methyl groups provide a tag
In mammals always part of a
symmetrical sequence
Concentrated in CG-rich
domains
Often in promoter regions
Methylation of promoter DNA
highly correlated with gene
repression
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DNA Methylation
Maintains a gene in inactive state rather than
initiating gene repression – Example:
Inactivation of genes of one X chromosome in
female mammals occurs prior to a wave of
methylation
Shifts throughout life in DNA-methylation
levels
Early Zygote – most methylation tags removed
Implantation – a new wave of methylation occurs
Important example – Genomic Imprinting
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DNA Methylation
Genomic Imprinting
Certain genes are active or inactive during early
development
Depending on whether they are paternal or maternal
genes
e. g.– IGF-2 is only active in the gene from the male
parent
The gene is imprinted according to parental origin
Mammalian genome has > 100 imprinted genes in
clusters
Imprinted due to selective methylation of one of the
alleles
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DNA Methylation
Genomic Imprinting
In the early embryo the waves of demethylation
and new methylation do not affect the
methylation of imprinted genes
Thus the same alleles are affected in the zygote
through to the adult stage in the individual
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Chromatin structure and
transcription
DNA is not naked – but wrapped around histone
complexes to form nucleosomes
How are transcription factors and RNA polymerases
able to interact with DNA tightly associated with
histones?
Apparently nucleosome structure does inhibit initiation of
transcription
Initiation of transcription requires assembly of large
complexes and nucleosomes block assembly at the core
promoter
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Role of Acetylation
Genes which are actively transcribed are bound
by histones which are acetylated
Each of the histones has a flexible N-terminal
tail
Extends outside the core particle and the DNA helix
Acetyl groups are added to lysine residues by
enzymes
Histone acetyl transferases (HATs)
Acetylation has two functions
Neutralize the positive charge on the lysine residues
Destabilize interactions between histone tails and
structural proteins
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Role of Acetylation
Some coactivators have HAT activity
Links histone acetylation, chromatin structure and
gene activation
HAT activity of coactivator acetylates core
histones bound to promoter DNA causing
release of nucleosome core particles or loosening of
histone-DNA interaction
Subsequent binding of transcription factors and RNA
polymerase
Once transcription is initiated – RNA polymerase is
able to transcribe DNA packaged into nucleosomes
Acetylation is dynamic – enzymes also remove
acetyl groups
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Role of Deacetylation
Removal of acetyl groups
Histone deacetylases (HDACs)
HDACs associated with transcriptional
repression
HDACs are subunits of larger complexes –
corepressors
HDACs guided to regions of DNA by methylation
patterns
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Role of Deacetylation
Example:
Inactive X chromosome of female
Largely deacetylated histones
Active X chromosome has a normal level of
histone acetylation
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Control of Acetylation / Deacetylation
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Control of Acetylation / Deacetylation
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Processing-Level Control
Recall that the formation of multigene families is
a mechanism that generates protein diversity
Protein diversity also generated via alternate
splicing
Regulates gene expression at the level of RNA processing
A mechanism by which a single gene can encode two or more
related proteins
Most genes (and their primary transcripts)
contain multiple introns and exons
Often – more than one pathway for processing of primary
transcript
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Processing-Level Control
Transcripts from approx 35% of human genes
may be subjected to alternate splicing
Simplest case – a specific segment either
spliced out or retained – Example:
Fibronectin:
Synthesized by fibroblasts – two additional
peptides compared to that synthesized by liver
Extra peptides encoded by pre-mRNA retained
in fibroblast
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Translational-Level Control
Wide variety of mechanisms – affecting
mRNA previously transported from the
nucleus
Subjects include:
Localization of mRNA in the cell
mRNA translation
Half-life of mRNA
Mediated via interactions between mRNA and
cytosolic proteins
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Translational-Level Control
mRNA noncoding segments – untranslated
regions (UTRs)
5’ – UTR – from methylguanosine cap to AUG
initiation codon
3’ – UTR – from termination codon to end of
poly(A) tail
UTRs contain nucleotide sequences which
mediate translational-level control
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Translational-Level Control
Cytoplasmic localization of mRNAs –
Example ferritin
Translation regulated by iron regulatory protein
(IRP)
Activity of IRP dependent on cellular iron
concentration
At low iron concentration – IRP binds the 5’
UTR
Bound IRP interferes physically with the
binding of a ribosome to the 5’ end of the
mRNA
At high iron concentration the IRP changes
conformation and looses affinity for the 5’ UTR
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Control of mRNA stability
Half-life of mRNA is variable – 10 minutes to
24 hours
Specific mRNAs are recognized in the
cytoplasm and treated differentially
mRNAs lacking the poly(A) tail are rapidly
degraded
Poly(A) tail is not naked mRNA but bound by
the poly(A) binding protein (PABP)
Each PABP bound to about 30 adenosine residues
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Control of mRNA stability
PABP protects poly(A) tail from general
nuclease activity
But – increases its sensitivity to poly(A)
ribonuclease
mRNA in cytoplasm is gradually reduced in
length by poly(A) ribonuclease
When the tail is reduced to approx 30 residues
mRNA is rapidly degraded
Degradation occurs from the 5’ end
Suggests two ends held in close proximity
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