Control of Gene Expression Flashcards
what are recognition sequences?
recognition sites for DNA binding proteins
can be close or far away from gene
what are gene regulatory proteins?
transcription factors that will bind and activate genes
associate w/ the major groove of DNA structure
gene regulatory proteins and their binding locations
protein surface is completely complimentary to surface of DNA binding region
contact has 4 possible configurations of base pairs
w/ 10-20 interactions
parts of gene regulatory proteins
DNA binding module
activation module
dimerization module
regulatory module
what modules are always present vs. could be present on gene regulatory proteins?
always – DNA binding, activation modules
might – dimerization, regulatory modules
dimerization module
could be present
forms dimers w/ other protein subunits
regulatory module
could be present
regulate the transcription factor
evidence for transcription factors being modular
using experiments where we cut out portions of the gene regulatory proteins we discovered that they are modular
aka certain regions do a specific thing
what are the 4 different structural motifs?
helix turn helix
zinc finger motif
leucine zipper
helix loop helix
what is the most common structural motif?
helix turn helix
2 alpha helices connected by a short chain of amino acids
helix turn helix
helix turn helix
turned at fixed angle
-the longer helix has the recognition and dna binding modules
-dna side chains recognize dna binding site
zinc finger motif
- -a zn atom located at the base of a finger-like structure
- -zinc finger domains found in clusters
- -have multiple contact points w/ dna = stabilization
2 alpha helical strands that contact DNA like a clothespin
leucine zipper motif
leucine zipper
- -activation and dimerization modules overlap
- -forms hydrophobics interactions between 2 helices = zipper
- -leucine every 7 amino acids
helix loop helix
a short alpha helix chain connected by a loop to a second chain
- -existing as a homo or heterodimer
- -3 modules: dna binding, dimer, activation
3 ways to identify transcription factors
EMSA
affinity chromatography
CHIP
electrophoretic mobility shift assay
EMSA
a gel mobility shift assay that detects sequence specific DNA binding proteins using a radioactive DNA fragment
after running a electrophoretic gel – the protein w/ smallest protein moves farthest – backpack example
affinity chromatography
to ID DNA binding proteins = purification
- ID a dna binding protein
- isolate
- use only 1 promoter recognition sequence
chromatin immuno-precipitation
CHIP
used when you don’t know what the regulatory protein binds to
–allowing you to ID sites in genome that a known protein binds to
CHIP use
used in living cells
PCR product at end so you can ID DNA sequence
how does histone acetylation effect binding to DNA
makes it easier to remove histones
thus easier access to DNA
activator and repressor protein competition
their binding sites might overlap and they can compete for binding
activator and repressor proteins both bind DNA
repressor can then bind to the activator to inhibit transcription = masking
or
repressor can bind to other DNA to block assembly of transcription machinery
what other factors can a repressor attract?
- -chromatin remodeling complexes
- -histone deacetylase
- -histone methyl transferase
chromatin remodeling complex recruitment
repressor can recruit these to fold up DNA and make promoter site unavailable
histone deacetylase attraction to promoter
repressor proteins can attract this which results in
it is harder to remove/remodel deacetylated histones – thus harder to free DNA from it’s coiled structure
histone methyl transferase to ______ histones
this will methylate histones
recruited by repressor proteins
resulting in other proteins binding to methylated histones keeping DNA transcriptionally silent
list the ways repressor proteins can stop transcription
- bind activator/DNA
- block assembly of machinery
- attraction of other factors
gene regulatory proteins acting as a committee
the same protein can act as a activator or repressor depending on what proteins have assembled together
aka = committee of proteins together will = an activator or repressor
how is the function of gene regulatory proteins regulated?
- synthesis
- ligand binding
- covalent modification
- addition of subunit
- unmasking
- nuclear entry
- proteolysis
synthesis
regulatory proteins are only made when wanted
ligand binding
regulatory proteins can’t work until a ligand binds
covalent modification
–phosphorylation
proteins are not active until modified
unmasking
an inhibitor is bound normally but once removed = activation
nuclear entry
regulatory proteins are only active if located w/in nucleus
proteolysis
the regulatory protein is normally membrane bound
becomes active when released from membrane
RNA alternative splicing - activators vs. repressors
activators can recruit splicing machinery
repressors can stop them from being able to bind
RNA modifications
these provide stability
poly A tail
5’ cap
iron excess responses
ferritin made
no transferrin receptor made
thus unable to uptake Fe into cells
iron starvation response
no ferritin made
transferrin receptors made
thus Fe is taken into cells
IRE’s
iron responsive elements
recognition sites for binding
when in iron excess
IRP
iron responsive regulatory protein
when in iron starvation
binding of IRPs to block translation
to IRE at 5’ ferritin mRNA
= no ferritin made
= translation blocked
if this does not happen mRNA is made so ferritin is made
binding of IRPs to allow translation
to IRE at 3’ transferrin receptor mRNA
= transferrin made
= mRNA stable
if this does not happen RNA degrades and no transferrin receptor is made
thus no iron taken into cell
ferritin
is made when in excess of Fe
binds to receptors to make cells uptake iron for storage when excess iron
transferrin receptors
found on basal cell membranes
uptake iron into cells
normally cells store iron but if in starvation these need to be blocked so that iron is not stored inside cells
iron starvation: IRP/IRE
IRP binding to IRE of ferritin
no mRNA made
because we don’t need to store Fe
IRP binds to IRE at 3’
=transferrin made to collect more iron
iron excess: IRP/IRE
too much iron in blood - must store excess in cells
ferritin needed but not TfR
IRP binds to iron and is inactivated
- -ferritin made w/o suppression
- -w/o binding of IRP TO TfR IRE – no transferrin made
microRNA function
small, non-coding regulatory RNAs to regulate mRNA
22 nucs long
function to silence expression of specific mRNA targets
microRNA binding
bind to complementary sequences in the 3’ UT end of mRNA
will induce degradation or just block translation
microRNA targets for binding
have widespread binding sites and repress hundreds of mRNAs or effect an entire program
post-translational modification of proteins: why?
required for protein to be functional/active
protein post-translational modifications
- by protein kinases
- glycosylated
- bind to other subunits/partners
- modifying enzymes act on protein
proteins modified by protein kinases
phosphorylation
modifying enzymes that act on protein: example
Hsp
help refold proteins so that can function properly
activated at high temperatures
regulation of proteins by degradation
proteasome
removes and recycles misfolded/old proteins
proteins are flagged w/ ubiquitin on lysine side chains
interior of proteosomes
a hollow chamber w/ proteasomes inside
ATP dependent
recognizes proteins w/ ubiquitin flags
recycles ubiquitin after binding bad proteins
addition of ubiquitin
using E1 enzymes
to add ubiquitin to lysine side chains on protein
coordinated gene expression
genes coordinate their expression in response to needs of the cell
DNA methylation
dna can be regulated by proteins
dna can be covalently modified to induce activation/suppression
methylation can be inherited from parents
genomic imprinting
- -differential expression of genetic material depending on parent of origin
- -associated disorder prader willi syndrome
PWS
prader willi syndrome
caused by paternal deletion on chromosome 15
PWS clinical presentation
infantile hypotonia
poor suck-feeding issues
stage two - childhood obesity - uncontrollable eating - hyperphagia
mental and behavior problems
PWS inheritance
deletion expressed if missing from dad even if proper genes exist in DNA inherited from mom
due to genomic imprinting
random X chromosome inactivation
females are XX
but the body cannot have segments of the same chromosome active
so in some areas of body one X is turned off while somewhere else the opposite is true
it is completely random and complex
