Gene Expression & Regulation Flashcards
What are the steps in transcribing DNA?
Recognise start and get into position
Start moving
Know when to stop
What does TFII stand for?
Transcription factor for Pol II
Why are general transcription factors needed?
Help position RNA pol II at promoter
Aid in pulling apart strands of DNA
Help release RNA pol II to start elongation
What is TBP and what does it do?
The TATA binding protein, binds in minor groove and induces a kink into DNA
What are the steps to transcriptional initiation?
TFIID (TBP) binds TATA sequence
TFIIB recognises BRE element, helps with positioning
TFIIF stabilises interaction between Pol II and other factors (TBP) - helps attract TFIIE and TFIIH
TFIIE attracts and regulates TFIIH
TFIIH unwinds DNA, phosphorylates Pol II tail
What are the steps to promoter escape and elongation?
TFIIH - DNA helicase hydrolyses ATP & unwinds DNA
RNA Pol II synthesises short lengths of RNA
Period of abortive initiation
Shifts to elongation when phosphate groups added to tail of RNA pol II
Disengages from GTFs
Acquires more proteins helping transcribe
Most of GTFs released to initiate another round of transcription
What are the 2 types of transcription factors?
General transcription factors
Activators/Repressors
What are activators?
Determine frequency of initiation/strength of promoter
Activators can have DNA binding activity, whilst
contacting other basal transcription machinery or
co-activators
Activators can have DNA binding activity whilst contacting GTFs/other co-activators
Some components of the transcriptional apparatus
work by changing chromatin structure
What are some mechanisms of activator action?
Recruit histone acetylase -opens up chromatin
Recruit chromatin remodelling complex
What are some mechanisms of repressor action?
Competitive DNA binding
Masking activator surface
Direct interaction with GTFs
Recruitment of chromatin remodelling complex
Recruitment of histone deacetylases
Recruitment of histone methyl transferase
What is the pre-initiation complex (PIC)?
Mediator - mediates interactions between activators and GTFs
Binds pol II & initiation factors & can enhance recruitment of pol II to activator-bound DNA
Detected at activator bound regions of DNA
What are Type I interferons?
Important antiviral signalling pathway
Infection triggers >3 activators
These activate IFN-B, which is secreted by infected cells to
warn/program uninfected cells into antiviral state
Activators cooperatively bind to enhancer upstream of promoter = IFN-beta enhancesome
How does control downstream of the IFN-beta enhancesome work?
Interferon stimulated genes (ISGs) normally suppressed by: repressor, high nucleosome occupancy, repressive complex bound to methylated histones
Upon IFN induction, activators bind to promoters and enhancers
Recruit chromatin remodelling & transcriptional activating complexes
Accuracy effects outcome of infection
How does a DNA footprinting assay work?
Underlying concept: DNA bound to protein protected from chemical/enzymatic degredation
Synthesize/amplify DNA of interest
Label - radioactive/fluorescent
Use agents to cleave it - DNase/hydroxyl radical
Visualise resulting pattern
How does an electrophoretic mobility shift assay (EMSA) work?
Used to ID where DNA proteins bind
Assumption: protein binding change in mobility of DNA in gel
Need: purified protein, antibody to protein, synthetic DNA and labelling
How does Chromatin immuno-precipitation (ChIP)
Used to determine all regulatory sequences occupied by a transcription regulator
Precipitate DNA using antibodies against gene regulatory protein A
Reverse crosslinks and remove protein
Amplify precipitated DNA by PCR
What is the CTD tail and why is it important?
52 repeats of 7 amino acids
Scaffold for RNA proteins
Proteins activate RNA Pol II
What is the process of 5’ mRNA capping?
5’ triphosphate of primary transcript cleaved
A guanosine residue added via 5’ to 5’ linkage
Cap guanosine methylated
What are the functions of the 5’ cap?
Protects RNA from degradation
Promotes pre-mRNA splicing
Needed for export from the nucleus
Required for efficient translation
How do proteins recognise specific sequences in mRNA?
Through small RNA guide connected to protein that base pairs with target
OR
Protein interaction between amino acids or stacking interactions
What ribonucleoproteins (RNPs) are used in splicing?
U1, U2, U4, U5, U6
Called sRNPs
Recognise 5’ splice site
Help catalyse RNA cleavage and joining reactions
What is the splicing mechanism?
U1 recognises 5’ splice site
BBP & U2AF bing branch point and splice site
U2 displaces BBP and binds to branch point creating a bulge
U4, U5, U6 join complex, U6 displaces U1
U4 displaced - intron lariat and active site formed, catalysis happens
U5 brings 2 exons together, final reaction
Why is alternative splicing useful?
Exons can be left out producing variety of proteins
Can be constitutive - multiple proteins from one gene
Can also happen as a regulatory mechanism
90-95% multi-exon proteins alternatively spliced
How is it defined which exons should be joined?
Regulatory proteins
SR proteins bind to exons and contact splicing machinery (‘exonic splicing enhancers’ - ESEs)
hnRNP proteins bind to RNA - don’t contact splicing machinery (intronic/extronic splicing silencer - I/ESS
How is splicing regulated?
Repressor - sits on splicing site blocking it
Activator recruits splicing machinery
How can mutations change splicing?
Normal splice site altered to cause exon skipping
Normal splice site altered and another (cryptic) site used
Mutation causes new splice site
What are examples of disease caused by splicing defects?
STAT1 - leads to lethal bacterial & viral infections, mutation in splice site results in shorter mRNA
Spinal Muscular Atrophy (SMA) - exon skipping results in low SMA protein levels
What are microRNAs?
Class of non-coding RNA
Pol II transcripts that get processed in nucleus then cytoplasm
Acts as guide for protein complex to all mRNAs - influencing translation and stability
Can be produced from introns or exons - intronic miRNAs interact with splicesome
What is the process of cleavage and polyadenylation of RNA
CTD tail recruits enzymes necessary for polyadenylation - CPST & CstF
Specific sequence at end of gene triggers transfer of factors
Recruits other factors for cleavage and polyadenylation
Additional proteins assemble to cleave RNA
PolyA polymerase (PAP) adds ~200 amino acids - no template
As its synthesised polyA binding proteins assemble on it
What makes RNA polymerase stop transcribing?
Transcribes terminator sequence which is GU-rich or U-rich
Why do RNAs need to be degraded?
Quality control - some byproducts of transcription need to be removed & recycled, if not made correctly
Gene regulation - if never removed couldn’t be regulated, highly regulated mRNAs have short 1/2 lives
What are the basic enzymes used for RNA degradation?
Exonucelase - 5’-3’ and 3’-5’
Endonuclease - internal within a strand
What are examples of 5’-3’ exonucleases?
Xrn2 - major human exonuclease in nucleus, transcription termination of RNA pol II (degrades uncapped transcript), pre-mRNA degradation
Xrn1 - cytoplasmic exonuclease, mRNA degradation (decapping required)
What’s an example of a 3’-5’ exonuclease?
Core exosome - 6 subunits - Rrp6 and Rrp4 are active subunits, uses many different cofactors for different purposes, involvedin processing and degradation and control lifetime of mRNA
Which RNA needs to be destroyed?
The introns (spliced out need to be reused and recycled)
When does nuclear RNA degradation occur?
If capping doesn’t happen properly - remove RNA using Xrn2 5’-3’ exonuclease
Stalling during degradation
Splicing - need to degrade introns, 1st lariat debranched with Dbr1, degraded from both ends
Termination - torpedo mechanism using Xrn2
Export failure - decapping then degradation, Xrn2 & exosome
Cryptic unstable transcripts (CUTs) - mediated by exosome
When does cytoplasmic RNA degradation occur?
Nonsense mediated decay (NMD) - premature stop codon
Non stop decay (NSD) - requires translation to detect no stop codon
No go decay (NGD) -stalling during translation
Regulated decay
What happens in nonsense mediated decay (NMD)?
Premature stop codon - could be from intron retention/frameshift
1 + exon-junction complexes no displaced - these assembled on mRNA as a result of splicing at exon-exon binding
Recruit Upf proteins that coordinate decapping and deadenylating enzymes
Happens as 1st ribosome translates mRNA
What are the consequences of a premature stop codon?
Non functional protein
Formation of amyloid
Loss of regulatory function in regulatory region - can lead to cancer
What happens in non-stop mediated decay (NMD)?
No stop codon on mRNA
Ribosome translates through to polyA tail - many lysines
Ribosome stalls, bound by Ski7, stimulates dissociation, recruits exsome
Proteins with poly-lysine at C terminus degraded by protease
How does degradation of ‘‘proper” mRNAs occur?
mRNAs undergo progressive deadenylation at rates specific to each mRNA
Once polyA shortening nearly complete cap removed and rest of mRNA degraded by exonuclease (5’-3’)
What is the untranslated region (UTR)?
3’ UTR sequence can have impact on mRNA stability & encodes regulatory sequences
Specific proteins recognise elements in UTR
Specific RNPs also recognise binding sites in 3’UTRs
Many affect deadenylation
How does the 3’ UTR affect the stability of mRNA?
ARE (AU-rich element) -mediated mRNA stability regulation: binds HUR proteins that increase half life by competing with destabilising factors
Non-ARE-mediated mRNA stability regulation: recognition of regulatory elements stimulates mRNA deadenylation
miRNA-mediated decay: UTR contains miRNA target site, recognition by RISC stimulates mRNA deadenylation
What are miRNAs?
Non-coding RNA
Pol II transcript that are processed & bind to complimentary regions in mRNAs
Translational repression
mRNA decay
