L3 Gene Expression: Regulation Flashcards
key regulators of biological function in health and disease
- changing RNA and protein levels
gene expression is regulated when?
- at multiple steps
changes in gene expression underlie carcinogenesis
- mutations in regulatory genes, including genes involved in chromosome modification result in cellular dysfunction and can lead to cancer
- mutations in regulatory genes and chromatin modifiers can affect expression of many genes
epigenetics
- broadly - changes in chromatin state
at what levels can you affect gene regulation?
- transcriptional control
- RNA processing control
- RNA transport and localization control
- mRNA degradation control
- translational control
- protein activity control
transcriptional activation via enhancers requires what
- opening of chromatin to allow transcription factor access at promotor
enhancer binds TF activators through
- through the mediator complex
- interacts with general TFs and RNA pol II to position RNA pol II at the promoter and allows transcription to occur
chromatin as transcriptional control
- major aspect of transcriptional control is opening and closing of chromatin
enhancers
- interact with general TFs to control transcription
- promote polymerase mediated transcription - recruit Pol II to the promoter
- enhancer is the sequence of DNA within the gene
enhancers can control
- spatial
- temporal
- inducible (hormonal, cell signaling)
transcription factors classified according to
- presence or absence of DNA binding motif
- activity
- chromatin modification
presence or absence of DNA binding motif
- DNA binding factors
co-regulators
(interact with DNA binding TF’s but don’t bind DNA)
DNA binding motifs
- zinc finger
- homeobox
- basic helix loop helix
activity
- transcriptional activator
- transcriptional repressor
chromatin modification
- acetylene or deacetylase activity
- open or closed chromatin
modularity of transcription factors
- have multiple functional domains
DNA binding domain of TFs
- often recognize sequence-specific patterns of hydrogen bond donors and acceptors in the major groove of DNA
specificity of DNA binding
- dimerization or protein interaction domains
histone modification domains
- acetylene and deacetylase activities
ligand binding
- nuclear hormone receptors (steroids, toxins)
cis control elements
- eukaryotic transcription factors bind short consensus sequences
how to TFs bind to a DNA
- as a homodimer or heterodimer
- must be present in high concentrations
PXR:RXR nuclear receptor in absence of ligand
- PXR:RXR binds a corepressor of proteins
- transcription is off
- HDAC3 removes acetyl groups from histones - closes chromatin
- active repression as it sits on the enhancer and is actively repressing transcription
PXR:RXR nuclear receptor in presence of ligand
- PXR:RXR undergoes conformational change and binds a coactivator complex (HMT, HAT, NRA)
- turns transcription on
- HAT adds acetyl groups to histones - opens chromatin and allows polymerases to be recruited
glucocorticoid receptor
- example of enhancer driven transcription
- TF with a DNA binding domain, an activation domain, ligand-binding domain, and nuclear localization sequence
GR in absence of steroid
- GR present in the cytoplasm bound a heat shock protein
GR in presence of steroid (its ligand)
- ligand binds to GR and displaces the heat shock protein
- conformational change that causes the HSP to fall off
- nuclear localization signal has been unmasked
- GR forms a homodimer and translocates to the nucleus
- GR binds to DNA at GRE enhancer and activation domain TAD recruits coactivators
- GR coactivator complex binds general TFs and activates transcription
mediator
- large multiprotein complex that “mediates” the effects of enhancer binding to the promoter
- different subunits interact with activators, GTFs, and Pol II
- stabilizes the binding of Pol II and GTFs at the promoter
- DNA often loops to bring activators and mediator together
chromatin
- DNA packaged into nucleosomes
core particle
- octamer particle with two histones H3 H4 H2A and H2B proteins
- DNA wrapped around core
histone tails
- have tails that are accessible for modification and transcriptional function
Histone H1
- linker histone residing on DNA between nucleosomes
histone modifications
- acetylation
- HAT can relax or open chromatin
- HDAC results in closed chromatin
- methylation
- phosphorylation
- ubiquination
- can really affect whether chromatin is condensed or not
HAT
- adds acetyl groups
HDAC
- removes acetyl groups
HMT
- adds methyl groups
KDM
- removes methyl groups
chromatin remodeling
- open and close chromatin to allow to deny access of transcription factors
chromatin remodeling carried out by
- histone modifications
- ATP-dependent complexes that move or restructure nucleosomes
nucleosome unwrapping
- can unwrap DNA from nucleosome to allow DNA binding protein access
nucleosome mobilization
- can move nucleosome around to allow for binding for transcription
nucleosome ejection
- Can get rid of nucleosome
histone dimer exchange
- can exchange one of the histones for another hone with different modification
DNA methylation and transcriptional control
- promoter (Cpg) is unmethylated and gene can be transcribed
- promoter (Cpg) is methylated and gene is silenced
what methylates the CpG promoters?
- DNA methyltransferase
methylation importance in imprinting and cancer
- tumor suppressor gene CpGs can be methylated and silence
common insulator binding protein
- CTCF
insulators and enhancers
- can block an enhancer from activating an adjacent gene
insulators and repressors
- can block the spread or activation of repressive chromatin from silencing the gene activity
chromosomal domains
- insulator-binding protein joins two insulators to form a loop and isolates sites of active or inactive transcription
barrier sequence
- prevents spread of heterochromatin to adjacent genes
alternative splicing
- exon skipping
- intron retention
- alternative 5’ splice site
- alternative 3’ splice site
- mutually exclusive exons
exon skipping
- splice 1 and 2 together, but skip over exon 3
intron retention
- keep one of the introns
alternative 5’/3’ splice site
- start splice in different sites to form larger or smaller exon
mRNA editing
- some mRNAs change bases after transcription due to an editing mechanism
- occurs in cytoplasm
- only on specific mRNAs at defined sites; not widespread
transferrin
- blood protein that transports iron
transferrin receptor
- membrane protein that binds transferrin allowing iron to enter the cell
- control at level of mRNA stability
iron is low - transferrin
- mRNA transferrin levels are high
- iron regulatory proteins bind transferrin receptor mRNA and stabilize it to protect it from degradation
iron is high - transferrin
- mRNA levels are low
- iron regulatory proteins bind iron but do not bind iron regulatory elements.
- transferrin receptor mRNA is degraded
ferritin
- iron binding protein that stores iron for future use
- control at level of translation
- iron response element at 5’ end can block translation
cellular iron levels high - ferritin
- need to store iron
- ferritin levels are high
- iron binds to iron regulatory protein and prevents binding to iron response element
- translation of mRNA initiated
cellular iron levels low - ferritin
- no need to store iron
- iron response element bind to iron regulatory protein and blocks translation
microRNA control of translation
- miRNAs are not translated
- form a hairpin which is trimmed in the nucleus
- miRNA binds to RNAs with different effects depending on the extent of the sequence homology
Dicer
- further trims pre-miRNA followed by degradation of one strand by Argonaute and RISC
strong match with miRNA
- degrades mRNA
weak match with miRNA
- inhibits mRNA translation
P bodies
- processing bodies
- cytoplasmic sites of translationally repressed RNAs and RNA degradation proteins
- possible role in regulating RNA levels and translation
RNPs and storage droplets
- can form storage droplet by phase separation
regulation of translation by guanine exchange factors
- one response to stress condition is a reduction in protein synthesis
- stress results in phosphorylation of translation initiation factors - reducing protein synthesis
regulation by GEF process
- eIF2-GTP is regenerated by guanine exchange factors called eIF2B during translation
- phosphorylation of eIF2B-GDP by protein kinases renders eIF2 inactive but still binds and sequesters eIF2B so it cannot reinitiate protein synthesis
ubiquination result
- targets for degradation
SUMOylation result
- targets for degradation
phosphorylation result
- activity
acetylation result
- activity
methylation result
- activity
what happens to misfolded proteins?
- they are targeted for degradation by specific proteases
- phosphorylation allows degradation
how does degradation occur?
- occurs by the ubiquitin/proteasome system
ubiquitin/proteasome system?
- ubiquitin added to protein
- forms proteasome
- get two things
- peptide fragments
- later degraded further into amino acids
- released ubiquitin
- peptide fragments
E1
- ubiquitin activating enzyme
- adenylates ubiquitin and attaches it to a high energy thioester on the enzyme
- recognized E2 enzymes
E2
- ubiquitin conjugating enzyme
- receives activated ubiquitin from E1
- binds to specific E3 enzymes
E3
- ubiquitin ligase
- recognize specific proteins and transfers ubiquitin onto them
protein decay and the inflammatory response
- Inflammatory response is initiated by the NFkB transcription factor in response to inflammatory signals (TNFa, IL1)
- NFkB is sequestered in an inactive form by binding to IkB
- IkB is phosphorylated in response to signaling
- When the phosphates are added, it is now a target for ubiquination
- Creates an E3 binding site ➞ IkB is ubiquitinated and degraded
- Releases NFkB ➞ translocates into nucleus resulting in transcription of pro-inflammatory genes
• Initiation of inflammatory response
