Lecture 38: Eukaryotic Gene Regulation Flashcards
3 classes of transcirption factors
1) DNA Binding Proteins
2) mediator proteins
3) chromatin remodeling enzymes
Epigenetic changes
alter gene expression by changing access of transcription factors to DNA sequences
histone modification
DNA Methylation
HATS (histone acetyl transferases)
faciliate gene activation
HDACs (histone deactylases)
inhibit gene expression
steroid receptors
function as ligand dependent and independent regulaturs that recruit HATs and HDACs
why remodel nucleosomes
they can block DNA binding to specific sequences
remodel in orer to premit gene expression
what is a nucleosome?
protein core of histones with DNA wrapped around it
unpacking chromatin structures
use HAT enzymes to add acetyl groups
open up the nucleosomal structures
repackaging DNA into chromatins
remove acetyl groups with HDAC
if chromatin condensed, chromatin
OFF
deacetylated
if chromatin decondensed, chromatin
ON
acetylated
epigenetics is what
chromatin modifications that alter transcription factor binding
acetylation or deacytlation by HATs and DHACs
what is the acetyl group donor in the HAT reaction
coenzyme A
what is the methyl group donor in the DNMT reaction
S-adenosylmethionine
How does acetylation change the structure of chromatin
pos charged lysine, acetylation takes away this pos charge, now its neutral
INTERACTS LESS WELL with NEG DNA since no longer pos charged
methylation in DNMT… how does this change the structure of DNA?
add methyl group to histone tail (when txn is ON)
to tigthen up and turn TXN OFF
Binding of chromatin remodeling complex (CRC) to tightly wound DNA
shifts postions of nucelosomes to make DNA binding sites accessible
3 types of sequence specific binding proteisn that chromatin remodeling uses
1)Basal transcption factors (likeTATA binding protein (TPB_)
not super specfic. bind to promoter to recruit Pol II
2) transcriptional actibator proteins
bind to sequence near promotorer and recruit HAT and CRC
3) transctiption repressor proteins
recruit HDAC and cytosine metholtransferase enzymes to repackage chromatin
TBP, a sequence speciic DNA binding protein
works at major groove,
recruits LOTS of other proteins to form complexes in order to get polymerase to right place
recognizing DNA sequences… helical elements
some side chains are really good at recognizing H bond patterns in DNA
mostly through interactions of heliciles in Major grove
3 protein structural motifs that insert helical elements into major groove
leucine zipper motif
zinc finger motif
homdeodomain motif
leucine zipper motif
has lots of leucines repeats for hydrophobic interactions that drive dimerization of 2 helicies
2 helicies (1 in front and 1 in back) that are inserted into major groove of DNA
part of larger protein complex
Do DNA binding proteins bind covalently or noncovalently to DNA? explain
noncovalently
easier to remove them, reversible, can go on or off depending on needs.
zinc finger motif
helix and loop are stablized by zinc ions
zinc finger are used to design DNA binding proteins that string the zinc fingers together so that you can target the sequence that you want
TAA USUALLY. Ts and As are important
homdeodomain motif
part that contacts DNA is small, makes contracts in major groove
specificity… finding what sequence a zinc finger prefers to bind to
give it a piece of DNA, randomize the sequence
see if it binds to it or not
free DNA smaller than bound DNA
gel shift assays
if theres a band, they say proteins will bind to that particular sequence
investigating comonallities of the binding
see that core has all Ts and As
some variability around that as you go further from the core
the core
what is found in every single position
enhancer sequences
bound by activator protein
can be near or far from TATA box
after bound by HAT,
recruit the other proteins,
mediator interacts with TBP and other transcription factors
TXN starts
where are enhancers
they can be far from start site because of looping out, they are eventually brought close to each other
Activator–> Mediator–>TBP–>???
RNA Pol
so what is bound
activator, mediator, TBP, recrut more transcription factors and RNA Pol 2, CTD stuff (poly A, splicesomes, capping stuff)
TRANSCRIPTION HAPPENS
Tissue specific Gene expression… how does this occur
by the state of chromatin packaging and presence/abseences of transcritption factors in different tussues
when are acetylation and cytosine methylation patterns established>
Early in development
affects expression of transcription factors in different cell types
“master” regulators
control expression of functionally related genes in tissues
Steroid receptors
great exampmple of eukaryotic gene regulation part of nuclear receptor family gluccocorticoid receptr estrogen receptor PPAR proteins retinoid receptors vitamin D receptors
what do steroid receptor proteins contain?
zinc finger DNA binding motif linked to binding domain with hydrophobic pocket
what are steriod receptors (activators or repressors)
both gene activators and repressors
depends on chem properties of the ligands and the gene sequence they bind to
what residues are important for binding to the zinc that stablizes the zinc finger structure?
cystine
FOUR models of gene regulation by nuclear receptors
1) Ligand-dependent direct activation
2) Ligand-dependent direct repression
3) Ligand-independent direct repression
4) Ligand-dependent indirect repression
Ligand-dependent direct activation
when nuclear receptors (dimers) are in presence of their ligand, they bind to DNA b4 start site
bind to gene and ligand results in recruiting HAT
HAT decondeses chromatin, TXN turned on
Ligand-dependent direct repression
nuclear receptor is associated with protein
ligand binding recruits HDAC
gene expression blocked
Ligand-independent direct repression
nuclear receptors are already bound to DNA
other factors don’t require direct binding of ligand to receptor, causes recruitment HDAC
blocked gene
RARE!!!!!
Ligand-dependent indirect repression
Ligand binds to nuclear receptor
Receptor does not have direct DNA binding, blocks gene expression in another way (perhaps interaction with other transcription factors)
What is the purpose of the nuclear receptor regulation?
whether HDATS or HATs will be recruited to turn off or on
What are mechanistic differences between prokaryotic/eukaryotic TXN factors?
EU: mostly by enzyme activity (HAT and HDAT). recruit enzymes for modification of chromatin
EU: interact more indirectly, don’t directly interact with RNA Pol, action at a distance
PRO: all about binding
