Regulatory Sequences In Eukaryotes Flashcards
Enhancers are?
Groups of regulatory sequences that control gene expression
Action at distance
When DNA Loops out to accommodate the interaction between proteins. This happens because some regulators bind far from the gene they control
Function of insulators
Block activation of promoter by activators bound at enhancers.
found between enhancers and promoters
What are activators?
They activate transcription by recruiting proteins to specific genes. They have DBD and AD
Example of activator in S cervisiae?
Gal4 activates transcription of galactose gene
Gal4 binds to?
4 sites located 275bp upstream of Gal1
Gal 4 activates the transcription of Gal1 in presence of?
Galactose
Domain swap experiment
Attaching AD of Gal4 to the DBD of LexA creates hybrid protein that activates transcription
What is a promoter?
Region at the gene where the transcriptional machinery binds
Helix- turn- helix motif consist of?
2 alpha helices separated by short turn.
The recognition helix fits in major groove. Other helix makes contact with DNA backbone
DBD motif that incorporate zinc?
Zinc fingers and zinc clusters.
In both cases zinc interact with cystine and histidine residues
Homeodomains are?
A type of helix-turn-helix motif.
consists of 3 alpha helices.
Helix 3 is the recognition helix.
Other 2 resemble HTH motif
Leucine zipper motifs are?
Made of 2 alpha helices that form a pincer like structure that grips DNA
each alpha helix is inserted into major groove half a turn apart
In leucine zipper motif dimerisation is mediated by?
Coiled- coil
Helix-loop-helix motifs:
An extended alpha helical region from each of the two monomers inserts into the major groove.
Similar how to leucine zipper works
Dimerisation in HLH motif caused by?
Interactions of recognition helix and shorter helix.
Dimerisation surface of HLH motif formed of?
Two helical regions. The first is part of the helix involved in recognition, the other is a shorter helix.
These two helices are separated by a flexible loop
In eukaryotes activators rarely interact with?
Polymerase
Eukaryotic activators recruit ………….. Indirectly and other ……………. Needed after polymerase has bound
Polymerase, factors
Functions of activators are?
Interact with: 1.transcription factors 2. chromatin modifying proteins 3. factors involved in elongation and initiation May recruit: 1. individual element TFIID or mediator 2. pre initiation holoenzyme
Activators recuit all ……………. To the …………..
Elements, promoter
Activators interact with parts of ……………. …………….. Other than………….
Transcriptional machinery , polymerase
TFIID is?
Transcription factor for polymerase II
TATA is recognised by
TFIID
Activator bypass experiment?
Gal1 gene activated in absence of Gal4
by fusion of DBD of LexA to a component of the mediator complex Gal11
In chromatin modification activators recruit…………… …………….. To help activate a gene packed within………………..
Nucleosome modifiers
Chromatin
Types of nucleosome modifiers?
- Histone acetyltranferases add acetyl group to tails of histones
- SWI/SNF displace nucleosome to remodel chromatin
Chromatin modification results in?
- Uncovering of DNA sequence
- Mobility of nucleosome to free up binding sites
- Addition of acetyl groups to histones tails alters interaction between tails and adjacent nucleosomes
Adding acetyl groups also help transcriptional machinery because?
It creates binding sites on nucleosomes for proteins bearing called Bromodomains
A components of TFIID complex bears………………. And also binds ………………… …………………. Better
Bromodomains
Acetylated nucleosomes
hps70 activated by ……….. ……………
Heat shock
GAGA factor believed to recruit enough of the ……………… ……….. To the gene for ………….
Transcriptional machinery
Initiation
Non heat shock conditions
- GAGA factor causes chromatin remodelling and initiate transcription
- RNA polymerase II stalls afters 20-50 nucleotides
Under heat shock conditions
- HSF binds at promoter and interacts with mediator
2. HSF recruits P-TEF which phosphorylates RNA polymerase II allowing transcription to continue
HSF is……………….. ………… ………….
P-TEF is……………. ………. ………… ………
Heat shock factor
Positive transcription elongation factor
HIV TAT recognises a specific sequence near the start of ……. ……… And present in the transcript made by stalled…………….
HIV RNA
Polymerase
On domain of ……… interacts with ………… And recruits it to the stalled………..
TAT
PTEF
polymerase
Activator SP1 initiates ……………. From ………… …………
Transcription
HIV promoter
Polymerase………… Having only produced a small amount of………… (……… ……….)
Stalls
RNA (TAR RNA)
………… Bind to …….. ………And recruits …………… Results in ………… Of RNA polymerase allowing transcription of ………. …….. To continue
TAT TAR RNA PTEF Phosphotylation HIV RNA
Models of action at distance?
Activator bind to enhancer and:
- may change DNA topology
- move along DNA to promoter
- Loop DNA to be closer to promoter
- loop DNA And increase size of loop until promoter reached
Insulators functions are?
- ENHANCER BLOCKING
Inhibit activation of gene by enhancer - BARRIER
Inhibit spread of chromatin modification
Insulators are placed ?
Between enhancer and promoter
Silencing is ?
A form of repression that can spread along chromatin switching off multiple genes
Drosophila insulator su(Hw)
- Two copies near each other.
NO INSULATION ACTIVITY - Place between enhancer and promoter.
SOME INSULATION ACTIVITY - A copy placed on either side of enhancer. HIGHEST INSULATOR ACTIVITY
S. Cervisiae maturing type switching
- mother becomes a before dividing
- switching controlled by the presence of HO gene
HO protein is a ………….. ………….. Which triggers………….. ……….. By creating a …………. …………. Break
Specific endonuclease
Gene conversion
Double strand
HO is ……… In the mother cell and kept………… In the …………. …….. Because of selective repressor ……..
Activated
Silenced
Daughter cell
Ash1
SW15 protein responsible for activating expression of ……. ……. It’s present in the ………. …….. And …….. …….. During budding.
But it’s ability to turn on HO is restricted because of presence of ……… In daughter cell
HO gene
Mother cell
Daughter cell
Ash1
SBF induces expression of …….. Binds near ….. ……….. It’s only ………… At the correct ……….. Of ……… ………..
HO HO Promoter Active Stage Cell cycle
In Mother cell ?
- SW15 binding recruits a chromatin remodelling complex SWI/SNF and histone acetyltranferase SAGA
- Results in SBF binding site becoming accessible
- SBF recruits transcriptional machinery and HO gene is expressed
In daughter cell?
- SW15 binding results in the transcription of Ash1
- Ash1 binds to HO promoter preventing SBF from binding
- HO gene is not expressed
Combinatorial control S. Cervisiae mat locus
In a cells
- Mat-a encodes the transcription factor a1
- MCM1 binds to promoters of a-specific genes
- Alpha-specific genes are silent without alpha1 protein
Combinatorial control S. Cervisiae mat locus
In alpha cells
- Mat-alpha encodes for factors alpha1 and alpha2
- MCM1 binds alpha1 to promoter of alpha-specific genes and represses their transcription
- MCM1 binds to promoters of haploid-specific genes and activates transcription
Combinatorial control S. Cervisiae mat locus
In diploid cells a/alpha
- Mat-a expresses the a1 protein
- Mat-alpha expresses alpha2 protein
- A hetorodimer of a1/alpha2 represses transcription of alpha1 and all haploid specific genes
- MCM1/alpha2 complex represses a-specific genes
- Alpha specific genes are silent without alpha1 protein
MCM1 is a
Transcription factor; involved in cell-type-specific transcription
Transcriptional repressors. Ways of working:
A. competition for activator binding site
Repressor can inhibit binding of the activator to a gene, thus block activation of that gene
Transcriptional repressors. Ways of working:
B. Inhibition of the activator
Repressor binds to a site on DNA beside activator and interacts with that activator.
Transcriptional repressors. Ways of working:
C. Interaction with components of transcriptional machinery at promoter
Repressor interacts with transcriptional machinery at promoter
Transcriptional repressors. Ways of working:
D. Recruitment of nucleosome modifiers
- histone deacetylases: remove acetyl groups from histones
- histones methylases: add methyl groups to histones
Repressor is:
A regulatory protein that bind to an operator and blocks transcription of genes
Gal genes. Mig1 Repressor
- Mig1 binding site between UAs and promoter
- Mig1 binds to this site in presence of glucose
- Mig1 recruits repressor complex containing Tup1
- Tup1 interacts with transcriptional machinery to repress initiation and recruits histone deacetylases
- Alpha2 recruits this repressor complex to prevent expression of a-specific genes in alpha cells
Signal transduction
How does the signal induce regulator activation?
1. Unmaking of activator domain
- Gal4 bound to the UAs in absence of galactose
- Gal80 masks the Gal4 AD
- Gal80 also recruits histone deacetylases
- Galctose cause Gal 80 to release from Gal4 AD
Signal transduction
How does the signal induce regulator activation?
2. Movement of regulator from cytoplasm by inhibitor cactus
- Drosophila dorsal protein held in the cytoplasm by the inhibitor cactus
- Ligand binding and signal cascade results in phosphorylation and destruction of cactus
- Released dorsal enters nucleus and activates genes.
Signal transduction
How does the signal induce regulator activation?
3. Controlled destruction of regulator
- in absence of signal, regulator B-catenin is phosphorylated by GSK-3B, regulating ubiquitylation and destruction
- ligand binding to Wnt receptor inhibits GSK-3B activity. B-catenin not destroyed
- enters nucleus and activates gene
What is Coiled- coil?
where the two helices are held together by hydrophobic interactions between leucine residues
