Eukaryotic Gene Regulation Flashcards
RNA polymerases in eukaryotes?
Pol I - makes rRNA
Pol II- makes all mRNA
Pol III - makes different types of non coding RNAs
Importance of pol II C terminal domain?
Phosphorylated and dephosphorylated for regulatory reasons
RNA pol TFs?
Pol II relies on help from TFs
TFII - promoter - helps to position pol II at promoter and aid in pulling apart the dna strands
Helps release rna pol II to start elongation
TBP
TATA binding protein
Aka TFIID
Binds TATA sequence in minor groove and introduced kink into DNA (80 degree angle) to help initiation of transcription
Provides platform for pre-initiation complex
RNA pol II initiation process?
TBP/TFIID binds TATA
TFIIB then recognises the BRE element just before the TATA sequence -helps with positioning
TFIIF then stabilises jnteractions between pol II and other factors (TBP) - and attracts
TFIIE which attracts and regulates TFIIH
TFIIH unwinds the dna and phosphorylates pol II tail
What activity does TFIIH have?
Helicase
ATPase
Kinase
How die pre initiation complex start moving?
Promoter escape
Then elongation
After TFIIH hydrolyses ATP and unwinds the DNA
Pol II synthesises short lengths of RNA (abortive initiation)
Elongation - phosphates groups added to pol II tail
And once it starts moving the general TFs disengage and are released so can start initiation elsewhere
Initiation and chromatin?
Chromatin structure and activators and depressors that control it affect which genes can be bound by the GTFs
Another level of gene control
E.g. activators binding chromatin remodelling complex so that transcription machinery can access and bind dna
GTFs vs activators and repressors?
GTFs - where
A+R - when and how much
Activator properties?
Distinct from GTFs
Control frequency and how much transcription
Some work by changing chromatin structure
Co-activators do not bind dna directly
Can activate by:
Covalent histone modifications
Nucleosome removal
Histone replacement
Repressor properties?
Competitive dna binding:
Bind close to promoter and prevent activator action
Masking activation surface:
Binds not so close to activator but interacts with it in a way that blocks ability to recruit factors
Direct interaction with GTFs:
Bind and directly interact with factors so activator can’t do it itself
Recruit chromatin remodelling complexes:
Remodel Nucleosomes to make them less accessible
Recruit histone deacetylases:
Acetylation is activation marker
Recruit methyl transferases:
Methylation CAN confer inactivation
Pre initiation complex properties?
PIC
Has a mediator complex that mediates interaction between activators and machinery
Can enhance pol II recruitment to activator bound DNA
Interferon pathway?
Viral infection
Triggers activators
NFkB
IRF-3/7
Jun/ATF
These activate IFN B gene
Secreted by cells to stimulate uninfected cells into an antiviral state
Hard on cells so want to tightly regulate
How to determine what parts of dna are bound by certain proteins
ChIP (works for in Vivo too)
Allows seeing where certain types of regulatory markers/elements are
Footprinting/EMSA
DNA bound to protein is protected by degradation
Amplify dna of interest
Label it
Incubate with protein
Degrade dna with DNase or hydroxyl radical
Visualise resisting pattern in gel alongside sequencing ladder
Certain fragment(s) missing - footprint - sequence where the protein was bound
Electrophoresis mobility shift assay:
Protein bound to dna will affect its migration through gel
ncRNA?
Non coding rna
C terminal tail of rna pol II?
7 AA 52 repeat
Scaffold for rna processing proteins
Regulated by phosphorylation
5’ mRNA cap properties?
5’ triphosohate of primary transcript is cleaved
Guanosine residue is added via a 5’-5’ linkage
This cap Guanosine is methylated
mRNA will be degraded without a proper cap
Cap promotes pre mRNA splicing
Needed for export out of nucleus
Required for efficient translation (recruits ribosome to mRNA)
Cleavage and polyadenylation
Happens while mRNA is still bound to pol II
C terminal tail is specifically phosphorylated
Causes recruitment of
CPSF - cleavage and polyadenylation specificity facto
CstF - cleavage stimulation factor
Specific sequence at end of gene triggers these factors to be transferred to mRNA (polyA signal - also defines where the RNA is cut)
additional proteins then assemble to cleave the RNA
PolyA polymerase then adds 200 adenine residues 1 at a time with no template
PolyA binding proteins assemble on it as it is synthesised
How does RNA pol II know when to stop?
pol II can continue transcribing - however this rna has no 5’ cap so is degraded (torpedo model)
mRNA Splicing process
2 step catalytic process
involves formation of intron lariat
Adenine residue in intron
attacks the 5’ splice site of the intron through nucleophilic attack
creates lariat structure (5’ end of intron joined to A at middle of intron)
5’ exon is then joined to the 3’ exon on the other side of the intron leaving spliced exons and the intron lariat
- U1 recognises 5’ splice site
- BBP and U2AF bind branch point (ADenine residue) and 3’ splice site
- U2. displaces BBP, binds to branch point making the A bulge out
- U4,5,6 join complex, U6 displaces U1
- U5 brings the 2 flanking exons together
How are the 5’ and 3’ splice sites recognised?
specific sequence motifs recognised by snRNPs (small nuclear RNPs)
U1, U2, U4, U5, U6 RNA guides bound to protein recognise 5’ splice site and branch site
help catalyse RNA cleavage and joining reactions
ways that a protein can recognise an RNA sequence motif?
- through direct amino acid interactions and bases
- through a small associated rna guide that base pairs with the target (ribonucleoprotein complex, RNPs)
alternative splicing properties?
make >1 funtional rna from same gene
constitutive - gene locus always produces multiple mRNAs
can be regulatory - one cell type splices one way, another type splices another way
defining which exons should join together to give alternate transcripts?
regulatory proteins promote splicing at appropriate places
SR (serine and arginine rich) proteins bind exons and contact splicing machinery (ESEs - exonic splicing enhancers)
hnRNPs (heterogeneous RNPs) bind RNA but dont contact splicing machinery(intronic/exonic splicing silencer - E/ISS)
action of alternative splicing regulators?
regulation in cell specific way:
Cell 1 - does not express repressor
even though it has repressor site next to splice site
so splicing goes ahead ther
Cell 2 - expresses repressor so does bind repressor site blocking splicing machinery
same but other way around for activator
presence of given activator/repressor key for splicing activity
alternative splicing and UTRs?
alternative splicing does not only affect the OFR
alternative last exon -
UTR sequence comes after last exon
an alternative last exon may be behind that one with a different UTR behind it
so skipping that exon and going for the alternative last one gives a different (e.g. longer) UTR which can affect that RNA’s turnover
mutations and alternative splicing?
normal splice site altered - causes exon skipping as that exon is not cut off from the introns
normal splice site altered and another cryptic splice (sequence with potential for interacting with spliceosome) site is used - can e.g. extend an exon
mutation causing a new splice site
extra exons inserted between existing ones
other products of RNA pol II besides mRNA?
microRNAs
class of ncRNA
Pol II transcripts - processed through series of steps in nucleus then cytoplasm
end up with ~22 nt mature sequence that guides a protein complex to mRNAs
important for regulating mRNA translation and stability
microRNA processing?
transcribed - then form stem loop structure based on self complementarity
recognised by enzymes in nucleus (DROSHA, DGCRA) which cut the stem loop to produce smaller 60-70nt precursor RNA
exported to cytoplasm - further processed by DICER to 22nt RNA and on eof these strands is kept
because of the stem loop structure of the original transcript - it is still duplex RNA
this duplex now binds to Argonaut and one of the strands is kept in this RNP complex - forms and RNA induced silencing complex
the 22nt RNA guides the argonaut complex to mRNA (through bp complementarity) to regulate it
forms a RIS complex
can be created with RNA from introns and exons
when does processing of miRNAs occur?
in tandem with splicing
purpose of degrading RNAs?
homeostasis and quality control (some rnas are transcription by-products need to be removed and recycled, or if the RNA was not made properly)
gene regulation (need to get rid of RNA to turn of expression of protein)
enzymatic mechanisms of RNA degradation
exonuclease - degrades from ends
require removal of 5’ cap to recognise that end (5’-3’ activity)
for 3’-5’ activity - exosome - 6 subunit complex (Rrp6, Rrp44 active nucelases)
involved in processsing and degradation
recognises abberant RNAs in nucleus
endonuclease - cuts internally
Nuclear RNA quality control pathways?
most rna degraded in nucleus - either after or during synthesis
capping failure - degraded before elongation occurs
during splicing:
introns need to be degraded too by Dbr1 (after splicing, lariat is debranched and degraded)
after termination:
RNA transcript aborts (after cleavage of mRNA, “torpedo model”) - Rat1 chases polymerase, pol then released
export failure - decapping and degradation
RNA degradation in cytoplasm?
detecting aberrant RNA:
after ribosome binding:
-premature stop codon
nonsense mediated degradation
-no stop codon
non stop decay - when ribosome reads all the way to ribosome tail
-stalled ribosome
no go decay
3’-5’ decay:
polyA tail removed by Caf1-Ccr4-NOT and degradation by exosome
5’-3’ decay:
decapping by Dcp1-Dcp2 and degraded by Xrn1
nonsense mediated decay?
NMD
premature stop codon in mRNA - don’t wan early stop protein (nonsense mutation)
can happen in long introns that are retained (stop codon sequence UAA can be contained there)
recognised as abnormal STOP as normal splicing deposits an Exon junction complex by 5’ and 3’ splice sites - ribosome kicks them off as it reads
- if STOP codon is reached before EJC yet to be kocked
-leads to UPF protein recruitment
triggerd degradation (decapping, deadenylation - susceptible to exonulceases - Xrn1 and exosome)
prevents premature terminated protein from being synthesised
regulated degradation of functional mRNAs
protein only needed for certain time
need to degrade its mRNA after a bit
remove polyA tail with deadenylase
remove 5’ cap too - stimulated by PolyA shortening
then 5’ exonulceases and exosome
deadenyilation and decapping linked and mRNA bends so that polyA is in proximity of 5’ cap
PolyA binding proteins removed by deadenylation interact with cap causing decapping
regulated turnover?
mRNA half life (normally 15min-10hrs)
e.g. cytokines have short half lives and elements in their sequence that mean quick turnover
RNA binding proteins (including RNPs MicroRNA-RISC) bind to the 3’ UTR downstream of the STOP codon
regulate mRNA stability
stabilisation/destabilisation of mRNA via 3’ UTR
3’ UTR is where degradation regulation happens
ARE site mediated:
TTP can recognise AU rich elements on the UTR - promotes deadenylation and so degradation of mRNA
HUR binds the UTR and prevents TTP from binding - stabilising the mRNA
miRNA mediated:
RIS complex e.g. argonaut
proteins can recognise these sites directly by AA interactions with AU rich sites
or through RNA intermediate to complementarity UTR sites (RNPs, RIS complex containing argonaut protein and promotes deadenylation e.g.)
AU rich element properties
ARE - binding site
AUBP - AU binding protein
mRNAs with short half lives have AREs in their UTR - hence quick turnover
methods for studying half lives?
figuring out of downregulation of mRNA is at trasncriptional level or half life level
reporter assay:
can see if certain gene is still being transcribed by cloning its promoter to a reporter enzyme (e.g. luciferase, GFP, LacZ)
(can identify promoter constructs by going upstream of genes and causing mutations - see if that has an effect on promotion? - increased expression = repressive region was mutated, decreased = promoter mutated)
transfect this plasmid vector into cell
incubate for RNA and protein to be made
measure activity of reporter to see if gene is still being transcribed
can measure mRNA degradation activity by cloning the UTR regulatory elements onto plasmid vector downstream of constitutive promoter and reporter gene
then transfect this into cell and incubate
then stop trasncription with inhibitors (e.g. actinomycin D which intercalates DNA and blocks transcription and elongation)
then can measure how long the reporter activity lasts
as the mRNA is degraded the activity will drop
so can tell whether drop in gene expression is due to transcriptional repression, or is it is due to increased degradation of the mRNA
