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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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Synthesis of a protein involves discrete steps
Several levels at which control mechanisms work
Transcriptional control
RNA processing control
Translation control
Protein activity control
Control of Gene Expression
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Differential gene transcription – the major mechanism of selective protein synthesisGoverned by a large number of proteins known as transcription factorsTwo functional classes of transcription factors
General transcription factorsSpecific transcription factors
Transcriptional level control
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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
Specific transcription factors
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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
Specific Transcription Factors
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Closest upstream sequenceTATA 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 sequencesCAAT boxGC box
Promoter Structure
Core
promoter
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PEPCKTATA 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
Control of PEPCK Gene Expression
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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)
Activation of Transcription
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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
Activation of Transcription
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Activation of TranscriptionExpression 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
Enhancers
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Control of Gene ExpressionActivation of Transcription
Transcription factor
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Activation of TranscriptionEnhancers
Control of Gene Expression
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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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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 coactivatorCoactivators are large complexes with 15 to 20 subunits
Do not directly bind DNA
Interact with a range of transcription factors
Action of Transcription Factor
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Contain different domains which mediate the different functions – at least two domains
DNA-binding domain
Activation domain
Commonly form dimers
ExampleGlucocorticoid receptor
Binds DNA at the glucocorticoid response element (GRE)
Ligand-binding domain / DNA-binding domain / Activation domain
Structure of Transcription Factors
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GRE
A palindrome
Two-fold nature is important
Pairs of GR polypeptides bind to DNA forming dimers
Transcription Factors Binding Element
5’-AGAACAnnnTGTTCT-3’
3’-TCTTGTnnnACAAGA-5’
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Transcription factors belong to each of several classes based upon specific types of binding domains or motifsMany contain an -helix which is inserted into the DNA major groove
Recognizes the particular nucleotide sequence lining the grooveBinding between aa and DNA (including DNA backbone) via:
Van der Waals (hydrophobic) forcesIonic bondsAnd hydrogen bonds
Transcription Factor Motifs
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Transcription Factor MotifsCommon transcription factor motifs
Zinc finger
Helix-loop-helix
Leucine zipper
HMG box
Shared featureStructurally stable framework
Specific DNA recognizing sequences are correctly positioned
Control of Gene Expression
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Types of DNA binding proteins
DNA and RNA polymeraserepair enzymes
structural proteinstranscription factors
Types of DNA binding proteins
DNA and RNA polymeraserepair enzymes
structural proteinstranscription factors
DNA binding motifs
zinc fingersleucine zippershelix-turn-helixhelix-loop-helix
DNA binding motifs
zinc fingersleucine zippershelix-turn-helixhelix-loop-helix
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Zn ion coordinated to two cysteines and two histidines
Each contains multiple zinc finger domains
Zinc finger
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Two a helices separated by a loopOften preceded by a stretch of basic aa which interact with a specific nucleotide stringAlways occur as dimers
homodimersheterodimers
Helix-loop-helix (HLH)
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Leucines every seven aa along an -helix
All leucines face the same direction
Two -helices can zip together forming a coiled coil
Basic aa on opposite side of coils
Leucine zipper motif
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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
Repression of Transcription
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Binding to promoter elementsBlocking assembly of the preinitiation complex
Mechanisms of Transcription Repression
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Inhibiting binding or functioning of transcriptional activators
Mechanisms of Transcription Repression
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DNA MethylationMethyl groups may be attached to cytosine (C5 position)
Methyltransferases
Methyl groups provide a tag In mammals always part of a symmetrical sequenceConcentrated in CG-rich domains
Often in promoter regions
Methylation of promoter DNA highly correlated with gene repression
Repression of Transcription
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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
DNA Methylation
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Genomic ImprintingCertain 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
DNA Methylation
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Genomic ImprintingIn 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
DNA Methylation
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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
Chromatin structure and transcription
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Genes which are actively transcribed are bound by histones which are acetylatedEach of the histones has a flexible N-terminal tail
Extends outside the core particle and the DNA helixAcetyl groups are added to lysine residues by enzymes
Histone acetyl transferases (HATs)
Acetylation has two functionsNeutralize the positive charge on the lysine residuesDestabilize interactions between histone tails and structural proteins
Role of Acetylation
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Some coactivators have HAT activityLinks 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 interactionSubsequent binding of transcription factors and RNA polymeraseOnce transcription is initiated – RNA polymerase is able to transcribe DNA packaged into nucleosomes
Acetylation is dynamic – enzymes also remove acetyl groups
Role of Acetylation
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Removal of acetyl groupsHistone deacetylases (HDACs)
HDACs associated with transcriptional repression
HDACs are subunits of larger complexes – corepressors
HDACs guided to regions of DNA by methylation patterns
Role of Deacetylation
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Role of DeacetylationExample:
Inactive X chromosome of femaleLargely 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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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
Processing-Level Control
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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
Processing-Level Control
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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
Translational-Level Control
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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
Translational-Level Control
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Cytoplasmic localization of mRNAs – Example ferritin
Translation regulated by iron regulatory protein (IRP)
Activity of IRP dependent on cellular iron concentrationAt low iron concentration – IRP binds the 5’ UTRBound IRP interferes physically with the binding of a ribosome to the 5’ end of the mRNAAt high iron concentration the IRP changes conformation and looses affinity for the 5’ UTR
Translational-Level Control
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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
Control of mRNA stability
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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 residuesmRNA is rapidly degraded
Degradation occurs from the 5’ endSuggests two ends held in close proximity
Control of mRNA stability