Lecture 26 Flashcards
describe ON/OFF states of bacterial transcription
ground state = ON –> basal activity bc sigma factor can easily find promoter to bind
repressed state = OFF
describe ON/OFF states of eukaryotic transcription
ground state = OFF –> DNA is inaccessible
active state = ON –> DNA changes conformation to be accessible
2 types of chromatin structure
- heterochromatin
- euchromatin
what is heterochromatin?
tightly packed nucleosomes/CLOSED DNA
where do we find heterochromatin?
structural DNA and non-transcribed regions (centromeres, telomeres, repetitive elements)
what is euchromatin?
loosely packed nucleosomes/OPEN DNA
where do we find euchromatin
transcribed regions
2 general components of transcription initiation
- cis-acting DNA sequences
- trans-acting proteins
3 examples of cis-acting DNA sequences
- promoter
- promoter-proximal elements
- enhancers/silencers
4 examples of trans-acting proteins
- general TF
- common TF
- cell/tissue-specific TF
- transcription co-factors
general TF vs common TF
general TF is for most genes, common TF is for some genes
what are transcription co-factors?
no DNA-binding domain –> relies on binding of other TFs
where do general TF bind?
core promoter upstream of protein-coding genes
what is an enhancer?
DNA region with multiple TF binding sites
describe distance and position requirements of prokaryotic transcription
prokaryote has operator btwn promoter and gene –> has specific distance and position requirements
describe distance and position requirements of eukaryotic transcription
in eukaryotes, TFs can act anywhere in the gene so there are no distance/position requirements –> relies on distance-independent cis-acting DNA sequences
where are distance-independent cis-acting DNA sequences located?
- can be close or far from TSS
- can be upstream/downstream/within a gene (introns)
i.e. ANYWHERE!
2 types of distance-independent cis-acting DNA sequences
- enhancer
- silencer
what is the role of enhancers?
promote transcription
what is the role of silencers?
prevent transcription
what do TFs influence? how?
can influence transcription and chromatin structure directly or indirectly
how are TFs expressed in an organism?
cell/tissue/time-specific manner
what is an enhanceosome?
sum of all proteins interacting DNA to bring far away regions close together and allow RNA pol to bind
4 domains of TFs
- DNA-binding domain
- dimerization domain
- ligand-binding domain
- activation/repression domain
difference btwn eukaryotic and prokaryotic TFs
prokaryotic TFs don’t have the distinct domains
describe the TF DNA-binding domain
unique to a TF
why do TFs have a dimerization domain?
most TFs are dimers
why do TFs have a ligand-binding domain?
acts as a sensor and determines whether TF can bind DNA
why do TFs have an activation/repression domain?
mediates interactions with other transcription machinery
difference between TFs and transcription co-factors
transcription co-factors have similar domains but no DNA-binding domain
2 types of transcription co-factors
- corepressors
- coactivators
purpose of reporter gene assay
link reporter gene (GFP, B-gal) with sequence of DNA to see if it has enhancer activity
describe the reporter gene assay results of Pax6 enhancer
every cell has the gene but pancreatic and lens cells have TF that binds enhancer to allow gene expression
why don’t different cells and tissues just have different genes rather than having cell/tissue-specific expression of 1 gene?
more efficient to have 1 coding sequence and just use different enhancers/silencers to change expression pattern
how does yeast use galactose?
yeast converts galactose to glucose for energy and carbon metabolism
how does yeast induce enzyme production for galactose metabolism?
enzymes involved are only produced in presence of galactose
4 galactose-dependent enzymes
- Gal1
- Gal2
- Gal7
- Gal10
3 regulatory enzymes in yeast gal system
- Gal3
- Gal4
- Gal80
what does Gal4 do in general?
has DNA binding domain that binds UAS enhancers upstream of Gal1/2/7/10 to induce their transcription
is the yeast gal system an operon?
no, each Gal gene has its own promoter
what happens in Gal4- cells? What does this mean about the role of Gal4?
gal enzymes uninducible by galactose
Gal4 is required for activation
what happens in Gal80- cells? What does this mean about the role of Gal80?
gal enzymes constitutive
Gal80 is part of repression
what happens in Gal3- cells? what does this mean about the role of Gal3?
gal enzymes uninducible by galactose
Gal3 is part of activation
describe the activation of Gal4
- Gal4 binds UAS but is inactive bc Gal80 binds its activation domain
- Galactose and Gal3 bind
- ATP changes Gal3 structure so it can bind Gal80 and remove it from Gal4
- Gal4 is active and can drive transcription
what is the role of Gal3?
Sensor - responds to galactose
Inducer - binds Gal80 to remove it from Gal4
how does GAL4 induce transcription?
activation domain of Gal4 binds proteins in transcriptional machinery
- recruits RNA pol II
- recruits chromatin modifying proteins
diff between TFs binding enhancer vs promoter
enhancer binds common TFs, promoter binds general TFs
what is TFIID?
general TF that binds TATA binding protein
what is the role of TATA binding protein?
positions TFs at TATA box (TSS) to get RNA pol to correct gene
what is the role of insulators?
as a complex, binds DNA sequences to organize DNA into TADs
why are TADs required?
to ensure that enhancer works on specific gene –> enhancer only works on a gene that is in the same TAD
example of a chromatin remodelling protein complex
Swi/Snf
describe the Swi/Snf system
uses energy from ATP hydrolysis and moves nucleosomes to expose binding sites, like TATA box at promoter so it can form enhancesome
is Swi/Snf an activator or coactivator? why?
coactivator –> doesn’t bind DNA but gets recruited by enhancer-bound TFs
