Lecture 4 - RNA Polymerase, Transcription And Regulation Of Initiation Flashcards
What are the 3 RNA polymerases?
Polymerase 1, polymerase II and polymerase III
What do polymerases do?
They transcribe
What are the core 5 subunits and are these conserved?
Beta’, beta, alpha 1, alpha, and w
What are the steps of transcribing DNA?
Recognise the start and get into position
Start moving
Know when to stop
The TFII is found in the pol II promoter. What does it do?
Transcribe factor for pol II
What does TFIIB do?
It’s needed at nearly all Promotors of Pol II
What does TFIIF do?
Help to position RNA poly II at promoter
What does TFIIE do?
Aid in pulling apart two strands of DNA
What does TFIIH do?
Help to release RBA pol II to start elongation
What is the TBT?
TATA binding protein
What does the TBP do?
Bind the TATA sequence in minor grooves of DNA causing a kink in the DNA. This is the major sequence specific protein-DNA interaction during initiation
What is the sequence of transcriptional initiation?
TFIID (TBP) binds TATA sequence
TFIIB - recognises BRE element which helps with positioning
TFIIF - stabilises interaction between polII and other factors (TBP) - helps attract TFIIE and TFIIH
TFIIE - attracts and regulates TFIIH
TFIIH - unwinds DBA m, phosphorylase’s Pol II tails (DNA helipads and ATPase activity, kinase activity)
After initiation how does elongation occur after DNA unwinds?
RNA pol II synthesises short lengths of RNA
Period of abortive initiation
Shifts ti elongation when phosphate group are added ti tail of RNA poly II - CTD
Disengages from GTFs
Acquires more proteins belong transcribe
Most of GTFs are released available to initiate another round of transcription
Activators - what does the basal apparatus do?
Determines the start point for transcription
What do activators determine?
The frequency of initiation/strength of promotor
What do activators have with DNA
Binding activity
What do activators contact?
Other basal transcription machinery or commercial-activators
How do some components of the transcriptional apparatus work?
By changing chromatin structure
What are examples of changing chromatic structure?
Covalent history modification
Nucleosome remodelling
Nucleosome removal
Histone replacement
There is different modes of Repression - what is the competitive DNA binding mode of action?
Activator binds near the repressor stopping the bringing which triggers the activator
There is different modes of Repression - how does masking the activation surface work?
Blocks the activator to bring and roaming to factors
There is different modes of Repression - how does direct interaction with the general transcription factors work?
This could directly bind to factors
There is different modes of Repression - How could the DNA become less accessible in order to stop the activation?
Remodelling the Nucleosome so the promotor cannot be reached this is fine through recruiting histones deacetylase or Histone methyl transferase.
How do activators open up chromatin to make the dna more accessible?
Bind to specific elements of promotors and recruit Histone acetylase which lets transcription factors bind to promotors
What do mediators do?
They mediate transcriptional activators and Pol II
What does the mediator complex do?
Bind pol II and it’s initiation factors and can enhance recruitment of pol II ti activator bound DNA in vivo
What are the infection triggers for type 1 interferons?
NFkB
IRF-3/7
Jun/ATF
What do the infection triggers of type 1 interferons do?
Activate IFN-B which is secreted by infected cells to warn/program y infected cells in an antiviral state
Where do activators bind?
Upstream of the TATA box in a place called the enhancesome
What method do you use to detect in vitro regions of DBA bound to protein?
Foot printing/ EMSA
What method do you use to identify regulatory proteins bound in vivo?
ChIP
How do you do DNA foot printing?
Synthesise or amplify DBA if interest
Label
Incubate with a protein
Use DNase or hydroxyl radical to cleave it
Visualise the resulting pattern along side a sequencing ladder
How do we identify where DNA binding proteins bind?
DNA binding changes mobility of dna on gel
You need purified protein and an antibody
What does ChIP determine?
Determine all regulatory sequences occupied by a given transcription regulator
Can be used to determine positions along genome bound by modified histones
How do you do a ChIP?
Get a protein in vivo and cross link protein to the DNA so it’s covalent attached.
Then lyse and break the the DNA. You then precipitate the DNA and use the antibody to get rid of the cords link and see what you brought down
You can do this through a PCR
Why add chemicals to DNA?
To get rid of the proteins protecting it
