Week 5 - Control of Gene Expression Part III and IV Flashcards
Control of transcription initiation
• used for most genes
• local structure of the gene is changed
(chromatin unwound, histones)
• general transcription apparatus binds to promoter
RNA is modified and processed
- can control expression of alternative products from gene
- mRNA is exported from nucleus to cytoplasm
- mRNA is translated and degraded
Eukaryotic gene expression is usually controlled
at the level of initiation of transcription
• by opening the chromatin
Activator/repressor proteins interact with specific
promoter elements
Activator/repressor proteins interact with specific promoter elements
• transcription activator/repressor proteins have at least 2 independently folded and distinct functional domains
– DNA-binding domain = makes sequence specific contacts with the control elements in the regulatory promoter or enhancer
– activation/repression domain is left free to recruit/bind various components of the transcription machinery to alter chromatin structure around the transcriptional start site, in order to activate transcription
• additional domains include: dimerization domains, ligand binding domains
Transcription activator/repressor proteins have at least 2 independently folded and distinct functional domains
- DNA-binding domain
- activation/repression domain
DNA-binding domain
(of transcription activator/repressor proteins)
makes sequence-specific contacts with the control elements in the regulatory promoter or enhancer
Activation/repression domain (of transcription activator/repressor proteins)
left free to recruit/bind various components of the transcription machinery to alter chromatin structure around the transcriptional start site - in order to activate transcription
A cell-based assay for determining
whether a transcription factor is an activator or a repressor
A cell-based assay for determining whether a transcription factor is an activator or a repressor
- transcriptional activators or repressors can be assayed for an ability to activate or repress transcription in an in vivo transfection assay
- system requires 2 plasmids
– 1 containing the putative transcriptional activator/repressor
– 1 containing a reporter gene and 1 or more binding sites for the protein
- both plasmids are transfected into cells at the same time and the production of the reporter gene mRNA (and protein) is measured
- the reporter gene often encodes green fluorescent protein for ease of assay
- useful for domains/truncated proteins to identify/map activator/repressor domains
Cell-based assay for determining whether a transcription factor is an activator/repressor
PICTURE
Domain-swapping experiments have identified
activation domains
Domain-swapping experiments have identified activation domains
- DNA binding on factor 1
- factor 2 has 3 regions - ABC
- combind DNA-binding domain of 1 with different regions of 2
- test on gene carrying binding site for factor 1
- see which of the[region2 + DNA-binding of 1] has gene activation
Domain-swapping experiments have identified activation domains
PICTURE
Mechanisms for transcriptional activation by activator proteins
- activation by recruitment
- activation by conformational change
- activation by altering chromatin structure
Mechanisms for transcriptional activation by activator proteins
ACTIVATION BY RECRUITMENT
activation domain interacts with 1 or more components of the transcriptional machinery and stabilizes its binding to the template DNA
Mechanisms for transcriptional activation by activator proteins
ACTIVATION BY CONFORMATIONAL CHANGE
activation domain induces a conformational change in the transcription machinery to stimulate RNA polymerase II to initiate transcription
Mechanisms for transcriptional activation by activator proteins
ACTIVATION BY ALTERING CHROMATIN STRUCTURE
activation domain recruits chromatin remodelling proteins (co-activators) to modulate chromatin structure around promoterse
Assembly of the preinitiation complex - steps
PICTURE
Activation factors bind sequences
upstream
Activation by interaction with basal transcription machinery
- activtion by recruitment - activation domain interacts with one or more components of the transcriptional machinery and stabilizes its binding to the template DNA
- increases binding of a particular component of the basal machinery so enhancing its assembly
- or, activator alters conformation of an already bound factor so stimulating the activity/stability of the complex
Activation by interaction with basal transcription machinery
PICTURE
Activation by interaction with basal transcriptional machinery
…
activators can interact with
TFIID
Activation by interaction with basal transcription machinery
Activators can interact with TFIID
- enhances binding of TFIID to TATA box, thereby improving trate of PIC assembly
- activators can alter conformation of TFIID so stimulating its activity by increasiing its ability to recruit other PIC comonents or by enhancing ability to stimulate transcription
- interaction is through TBP or one or more of the TBP-associated factors (TAFs)
- accessory proteins within TFIID talk between TFIID and transcription factor
Enhanced transcription via TFIID
PICTURE
Activation by interaction with basal transcription machinery
Interaction is through TBP or one or more of the TBP-associated factors (TAFs)
PICTURE
Activation by interaction with basal transcription machinery
Activators can interact with TFIID via
TAFs
- different activators taraget different TAFs
- different cell types can have cell type-specific TAFs
- TAFs are critical intermediates between activators and the basal transcription complex
- TFIIB (binds directly after TFIID) for RNA polymerase to escae
- TFIID –> enhanced fromation of other proteins binding –> affects binding of RNA polymerase
Activation by interaction with basal transcription machinery
Activators can interact with TFIIB
- allows stimulation of asembly of PIC/activity of basal transcriptional complex after TFIID has bound
- TFIIB interacts with acidic activators
- activators stimulate binding of TFIIB to the promoter and can also alter its configuration when bound, thus improving its ability to recruit other components of the PIC (such as RNA pol II)
TFIID and mediator
- TFIID interacts with TATA
- mediator - physical link between activator protein and RNA polymerase
Activation by interaction with basal transcription machinery
Activators can interact with TFIIB
PICTURE
Mediator complex is required for
activated transcription
Mediator complex is required for activated transcription
- activators interact with RNA pol II through the mediator complex
- CTD of RNA pol II interacts with the mediator complex
- this interaction is required for the response to transcriptional activators
- mediator forms a molecular bridge between activators and RNA pol Ii
Activators interact with RNA pol II through
the mediator complex
CTD of RNA pol II interacts with the mediator complex
• This interaction is required for the response to
transcriptional activators
Mediator forms a molecular bridge between
activators and RNA pol II
binds carboxy terminal of RNA pol II and bridges interactions with activators
The mediator complex
PICTURE
Structure of the mediator complex
• approximately 30 subunits comprisig 3 subcomplexes
- the head
- the middle
- the tail
- head interacts with RNA pol II CTD
- tail itneracts with activators
- different classes of activators interact with different mediator subunits
Mediator interacts with
- activator proteins
- Pol II transcription machinery
Mediator interacts with activator proteins and Pol II transcription machinery
Activators can interact with co-activators
- the principle that governs the function of all activators is that a DNA-binding domain determines specificity for the target promoter or enhancer
- the DNA-binding domain is responsible for localizing a transcription-activating domain in the proximity of the basal apparatus
- an activator that works directly has a DNA-binding domain and an activating domain
- an activator that does not have an activating domain may work by binding a co-activator that has an activating domain
The principle that governs the function of all activators is
a DNA-binding domain determines specificity for the target promoter or ehancer
• the DNA-binding domain is responsible for localizing a transcription-activating domain in the proximity of teh basal apparatus
The DNA-binding domain is responsible for
localizing a transcription-activating domain in the proximity of the basal apparatus
An activator that works DIRECTLY has
a DNA-binding domain and an activating domain
An activator that does not have an activating domain may work by
binding a co-activator that has an activating domain
• most work this way
Activators can interact with co-activators
- a co-activator does not bind DNA but acts to transmit the signal froom the DNA-bound transcriptional activator to the basal transcription complex
- CREB-binding protein (CBP/p300) - recruited to DNA by the transciption factor CREB
- also mediates transcriptional activation via a number of other DNA-binding transcription factors involved in a variety of signaling pathways
- CREB-binding protein (CBP) is a histone lysine acetyltransferase
CREB-binding protein (CBP/p300)
- recruited to DNA by the transcription factor CREB
- also mediates transcriptional activation via a number of other DNA-binding transcription factors involved in a variety of signalling pathways
- CBP is a histone lysine acetyltransferase
- doesn’t bnd DNA
- CREB = DNA-binding protein, activates transcription by recruiting CBP
- receives 8 different signals from 8 different DNA sites through this 1 coactivator
- affects structure of chromatin
CREB-binding protein (CBP/p300) is a
histone lysine acetyltransferase
The chromatin landscape
- nucleosome out the way, important sites between nucleosomes = transcription occurs (especially with CBP)
- acetylation removes nucleosomes (lysine)
Nucleosome
- the basic unit of DNA packaging in eukaryotes, consisting of a segment of DNA wound in sequence around eight histone protein cores
- 8 histone proteins - 4 types, 2 each
- every 300 bp
Accessibility to DNA is regulated by
chromatin structure
• heterochromatin = inaccessible, repressive
– no replication
– no transcription
– no DNA repair
Euchromaitn
accessible, extended structure
– replication
– transcription
– DNA repair
Histone post-translational modifications regulate
chromatin structure and function
Post-translational modifications of histones
- acetylation of lysine - activation
- methylation of lysine - activation, repression
- methylation of arginine - activation
Method
Activators can interact with co-activators
- CREB-binding protein (CBP) is a histone lysine acetyltransferase
- mediates transcriptional activation by changing chromatin structure directly (charge) or indirectly (recruitment of acetyl-lysine binding proteins)
- this allows access to DNA and transcriptional start sites etc by opening up chromatin
add acetyl to lysine (by CBP) amino acid group
–> no positive charge, electrostatic with DNA weak, loosens
Histone acetylation is associated with
transcription activation
Histone acetylation is associated with transcription activation
- lysine (K) acetyltransferaes (KAT) - an enzyme (typically present in large complexes) that acetylates lysine residues in histones (or other prtoteins)
- also known as histone acetyltransferase (HAT)
- HATs are often recruited to promoters by transcriptional activators in order to activate transcrition from the promoter by opening up/looseniing chromatin, or by recruitment of the basal transcription machinery
Acetylation
Activator-directed histone hyperacetylation
- GCN4 binds to upstream activating sequence (UAS) through DNA-binding domain
- GCN4 activatioin domain interacts with a multi-protein GCN5 HAT complex (co-activator) (GCN5 dragged along, modifies histone)
- this allows access of PIC to DNA and transcriptional start sites etc by opening up chromatin
(acetylation = histones slide out of the way –> gain access to TATA)
Reader of histone lysine acetylation
- acetyl-lysines are recognized by a family of binding proteins that contain bromodomains (BD)
- TAFII250 is part of TFIID and therefore transcription is promoted by the recruitment of the PIC
Acetyl-lysines are recognized by a family of binding proteins containing bromodomains
Bromodomain
Reader of histone lysine acetylation
Transcriptional activators can
remodel chromatin
Transcriptional activators can remodel chromatin
acetylation of histones weakens/loosens nucleosome which in turn can
- facilitate access of an activator to DNA
- recruitment of activators that recognize and bind to acetyl-lysine
- promote nucleosome displacement from promoter in an energy-dependent process
Transcriptional activators can remodel chromatin
PICTURE
Chromatin remodeling is an active process
- chromatin remodelling - the energy-dependent displacement or reorganization of nucleosomes that occurs in conjunction with activation of genes for transcription
- there are numerous ATP-dependent chromatin remodelling complexes that use energy provided by the hydrolysis of ATP
Activities of ATP-dependent chromatin remodeling complexes
- all remodeling complexes contain a related ATPase catalytic subunit and are grouped into subfamilies containingmore closely related ATPase subunits
- remodeling complexes can alter, slide, or displace nucleosomes
(slide –> sequence free between nucleosomes to bind)
(take off nucleosome = histone lost = gap)
(unwind around nucleosome)
- some remodeling complexes can exchange one histone for another in a nucleosome
- recruited by DNA binding protein (activator) = is a coactivator
•
Activities of ATP-dependent chromatin remodeling complexes
Nucleosome organization/content may be changed
at the promoter
Nucleosome organization/content may be changed at the promoter
- a remodeling complex does not itself have specificity for any particular target site, but must be recruited - is a coactivator
- remodeling complexes are recruited to promoters by sequence-specific activators
- the factor may be released once the remodeling complex has bound
- transcription activation often involves nucleosome displacement at the promoter
Nucleosome organization/content may be changed at the promoter
steps
- sequence-specific factor binds to DNA
- remodeling complex binds to site via factor
- remodeling complex displaces octamer
Nucleosome organization/content may be changed at the promoter
PICTURE
SWI-SNF chromatin remodelling complex
- a remodeling complex does not itself have specificity for any particular target site, but must be recruited in this case by transcriptional activator ‘X’
- SWI-SNF acts to atler chromatin structure of these genes thereby facilitating their subsequent activation by other transcription factors ‘Y’
- between nucleosomes is free
- DNA for factor X (transcriptional activator with only DNA binding domain) - needs transcription binding things
- SWI SNF hydrolyzes DNA, uncover 2nd DNA sequence for Y - one that can bind (y = transcription factor)
SWI-SNF chromatin remodelling complex
PICTURE
Transcriptional activation involves multiple changes to chromatin
PICTURE
Transcriptional activation is
synergistic/additive
Mechanisms of action of transcriptional repressors
- preventing an activator binding through chromatin structure
- overlapping binding sites
- repressor sequesters the activator
- repressor quenches the transcriptional ability of the activator
- repressor degrades the activator
- repressors can directly repress transcription
Mechanisms of action of transcriptional repressors
PICTURE
Transcriptional repression through chromatin structure
- act by establishing repressive/tightly packed chromatin (heterochromatin) around promoters
- acetylation of lysine = turns off
- methylation of lysine = activate and repress
- not a charged base effect
Transcriptional repression through chromatin structure
deacetylation of histones
- histone deacetylase (HDAC) - enzyme that removes acetyl groups from histones - may be associated with reperssors of transcription
- deacetylases are present in complex with repressor activity
Histone deacetylase (HDAC)
- enzyme that removes acetyl groups from histones
- may be associated with repressors of transcription
- remove lysine = repress
(turns back to positive lysine)
• repression ercruits this reverse reaction - removes acetyl
Deacetylases are present in
complexes with repressor activity
Transcriptional repression through chromatin structure
deacetylation of histones
PICTURE
Repressors can interact with co-repressors
- UME6 - repressor - binds to upstream repressor sequence (URS1) through DNA-binding domain
- UME6 repression domain interacts with a multi-protein SIN3 complex (co-repressor) that includes the RPD3 histone deacetylase
- this blocks access of PIC to DNA and transcriptional start sites etc by closing up chromatin
UME6
repressor binds to upstream repressor sequence (URS1) through DNA-binding domain
(repressors can interact with co-repressors)
UME6 repression domain can interact with a multi-protein
SIN3 complex (co-repressor) that includes RPD3 histone deacetylase
• this blocks acces of PIC to DNA and transcriptional start sties by closing up chromatin
Transcriptional repression through chromatin structure
methylation of histones
- methylation of lysine –> activation, repression
- repressors can modify chromatin structure through lysine methylation
- methylation is catalyzed by histone methyltransferases (KMTs)
Methylation
addition of CH3
must be regulated
Readers of lysine methylation
- methyl-lysines are recognized by a family of binding proteins containing chromodomains
- KMTs are ofter recruited to promoters by transcriptional activators and repressors in order to regulate transcription from the promoter and therefore co-repressors/activators
- 9th lysine methylation recognized by proteins (chromodomains) - mediate higher order and chromatin structure (wrap up histone very tightly)
Methyl-lysines are recognized by a family of binding proteins containing
chromodomains
KMTs are often recruited to promoters by
transcriptional activators and repressors
•in order to regulate transcription from the promoter and are therefore co-repressors/activators
Readers of lysine methylation
Spreading of heterochromatin
- HP1 binds to methyl-lysine on histone H3 Lysine 9 through its chromodomain
- following binding of HP1, it recruits a HMT which in turn methylates an adjacent nucleosome on histone H3
- this promotes spreading of the tightly packed chromatin structure and mediates trancriptional silencing
- reinforces itself
- corepressors calls forward another corepressor that methylates next histone…
Spreading of heterochromatin
PICTURE
Transcriptional repression by polycomb complexes
- during early embryogenesis repressors associated with PRC2 co-repressos complex
- resulting in methylation of histone H3 Lysine 27 by the E(z)
- PRC1 complex binds through its chromodomain to methylated lysine 27 and condenses chromain into an inactive form
- repession is maintained in the absence of the original repressor throughout adult development
- repress genes only needed in zygotic development (eg Hox genes)
- memory in mitosis of the repression
- repressor binds DNA at promoter
- PRC = corepressor
- Ez = histone methyltransferase of lysine27
- methylates K27 –> compress –> switch off gene
(binding site of polycomb complex PRC1 that switches it off forever)
Transcriptional repression by polycomb processes
Lysine methylation and acetylation crosstalk (H3K9)
- lysines can be acetylated or methylated
- by switching modifications, processes such as transcription can be regulated
Lysine methylation and acetylation crosstalk (H3K9)
PICTURE
Repressors can act indirectly by
inhibiting the positive effect of activators
Repressors can act indirectly by inhibiting the positive effect of activators
- repressor (R) binds to a specific DNA sequence preventing/blocking the action of the activator (A) or by binding to the activator itself
- repressor degrades the activator
• MDM2 is a repressor which ubquitinates the p53 activator, thereby promoting degradation by proteases
• AEBP1 is a repressor that is itself a protease and degrades the activator directly
Repressor (R) binds to a specific DNA sequence preventing/blocking the action of the activator (A) or by binding to the activator itself
MDM2
a repressor which ubiquitinates the p53 activator, thereby promoting degradation by proteases
p53 (tumor suppressor, regulates cell cycle)
- binds DNA, activates transcription
- regulated by accessory MDM2 that’s a repressor that changes p53 itself by modification of p53
• ubiquitin = protein degraded (by protease that’s recruited) –> p53 degraded
AEBP1
a repressor that is itself a protease and degrades the activator directly
Repressor degrades the activator
MDM2
AEBP1
Repressors can act directly by
inhibiting the assembly or activity of the basal transcription complex
Repressors can act directly by inhibiting the assembly or activity of the basal transcription complex
- repressor (R) binds to a specific DNA sequence and directly inhibits the activity of the basal transcriptional complex or by binding directly to the complex
- Dr1 factor binds TBP preventing binding of TFIIB and therefore assembly of the PIC
- Mot1 displaces TBP from DNA
- binding site for repressor/silencer in core promoter
- binding and repressor domains
- Dr1 = transcriptional repressor, binds TBP proteins - other TAFs and trancription factors don’t come
- Mot1 binds close to TATA = TBP can’t bind
Dr1 factor
binds TBP, preventing binding of TFIIB and therefore assembly of the PIC
binds TBP = other TAFs and transcription factors dont come
Mot1
displces TBP from DNA
(binds close to TATA = TBP can’t bind)
Repressor binds to a specific DA sequence and directly inhibits the activitiy of the basal transcription complex or by binding directly to the complex
Dr1 and Mot1
9th lysine in H3
- acetylation –> bromodomain –> loosen
- methylation –> chromodomain –> tighten
- can’t be both
- 1 amino acid = switch gene on/off
