- RNAP1 - 14 subunits, nucleolus, rRNA but not 5S rRNA.
- RNAP2 - 12 subunits, nucleoplasm, mRNA/snRNA/miRNA.
- RNAP3 - 17 subunits, nucleoplasm, 5S rRNA/tRNA.
- Each shares 5 small subunits and has their own unique subunits to confer unique properties.
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- TFs needed to direct Pols to gene promotors and allow recognition
- The main TF is TBP - a homodimer which binds the TATA box of promoters.
- It bends DNA 90 degrees.
- Part of the TFIID complex along with 13 TAFs.
- Also used at TATA-less promoters due to multiple recognition elements.
- TBP binds the TATA box.
- TAF1 binds inr.
- TAF6 binds the DPE.
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- TFIID is recruited and TBP binds TATA.
- TFIIA binds and stabilises this interaction.
- TFIIB is recruited and binds the BRE upstream and downstream of TATA, allowed by DNA bending.
- TFIIB recruits TFIIF in complex with Pol2.
- This recruits TFIIE which in turn recruits TFIIH.
- 10 subunits including XPB and XPD helicases and Cdk7 kinase/CAK.
- Cdk7 phosphorylates the Pol2 CTD on Ser2.
- Involved in DNA repair as well as transcription.
- Causes xeroderma pigmentosum when mutated in the XPB and XPD domains.
- This leads to extreme photosensitivity and defecting repair of UV-induced DNA damage.
- Leads to cancer predisposition and early death.
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- Pol1 and Pol3 have evolved attachement of TFIIE and TFIIF as subunits rather than TFs.
- Pol2 interacts more loosely with these at GTFs.
- Tight binding might improve transcriptional efficiency but it decreases regulatory flexibility.
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Open Complex Formation
- XPB helicase melts the dsDNA and feeds the template strand into the Pol2 catalytic centre.
- Pol2 scans for the TSS.
- TFIIB stabilises the open DNA conformation and helps in TSS recognition.
- Two NTPs bind opposite the template strand in the -1 and +1 positions allowing condensation.
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- Consists of repetitive condensation reactions to extend RNA by one NMP.
- 8 nucleotides of nascent RNA remain annealed to the DNA template strand in a 12 nucleotide transcription bubble.
- As the 3' end is extended, the 5' end peels away to maintain size of the bubble.
Active Site: Key Features
- Templating nucleotides form WC base pairs with incoming NTPs.
- 3' end of RNA stacks on the base of the incoming NTP.
- Asp residues co-ordinated two Mg2+ ions for catalysis.
- Only one Mg2+ remains in the active site, the other arrives and leaves with pyrophosphate.
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- NTP condensation is catalysed by two Mg2+ ions co-ordinated by Asp residues in the active centre.
- Mg2+ promotes deprotonation of the RNA 3'-OH.
- This leaves 3'-O- which attacks the α-phosphate of NTP.
- This forms a new phosphodiester bonds with release of pyrophosphate.
- This exits via an exit pore lined with a series of positively charged residues.
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Structure and Function
- The larges subunits of Pol2 (POLR2A) has a CTD made up of a heptad of repeats.
- It provides a docking platform for other proteins depending upon its phosphorylation state.
- Phosphorylation varies through the transcriptional cycle allowing dynamic recruitment of proteins.
- Pol2 is recruited to promoters with an unphosphorylated CTD.
- This binds to TBP to anchor itself to the PIC at the promoter.
- TFIIH Cdk7/CAK phosphorylates CTD on Ser5 during initiation leading to de-anchoring.
- During elongation Ser5 undergoes gradual dephosphorylation and Ser2 undergoes gradual phosphorylation.
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- Used to test which regions of DNA are bound by proteins in cells.
- Formaldehyde is used to crosslink proteins to nucleic acid (NA) in vivo.
- The chromatin is then isolated with bound proteins remaining where they were in vivo.
- The DNA is digested into small ~500bp fragments by sonication.
- This is incubated with an anti-protein antibody.
- The complex is precipitated using insoluble protein A agarose beads that bind the antibody and pellet the complex during centrifugation.
- Supernatant is removed and the pellet is washed several times in buffer.
- The sample is then heated to remove the cross links between protein and NA.
- The NAs are then characterised by PCR or sequencing.
- Bioinformatics is also used to align sequences against the reference genome.
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- Used to resolve proteins by approximate mass using SDS-PAGE.
- Run an SDS-PAGE gel with proteins and then blot gel onto a nitrocellulose membrane.
- Probe the membrane with protein specific antibodies.
- Wash away unbound antibody.
- Detect the bound antibody by addition of a secondary antibody that binds the primary antibody and is coupled to an enzyme that emits a signal when incubated with substrate.
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Phosphorylation and Recruitment
- mRNA capping enzymes bind the CTD with high Ser5-P.
- Ser5-P and Ser2-P recruit transription elongation factors and splicing factors.
- Low Ser5-P and high Ser2-P recruits mRNA 3' cleavage and polyadenylation factors.
- The SEC is a complex of proteins including P-TEFb, a kinase which phosphorylates Ser2 of CTD.
- In MLL, genes encoding subunits of the SEC become fused to the MLL1 gene by chromasomal translocation, resulting in aberrant transcription.
- DOT1L is an EF and HMT that performs H3K79me2 in Pol2 transcribed regions.
- It increases metastasis and invasion of lymph nodes by increasing migration of cells.
- siRNA knock-down of DOT1L suppresses migration and decreases motility of breast cancer cells
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- Most mRNAs end with AAUAAA polyadenylation sequences.
- It is recognised by RNA processing factors of Pol2 that cleave RNA and add a polyA tail for mRNA stability, translocation to cytoplasm and translation.
- Pol2 pauses downstream of this sequence and an exonuclease attacks the unprotected 5' end of RNA.
- This chews up the RNA and knocks Pol2 off the gene.
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- This is a toxin that inhibits Pol II.
- It is a cyclic octapeptide toxin produced by Amanita mushrooms.
- It binds the catalytic core of Pol II and blocks translocation along DNA.
- Cancer cells are especially sensitive to this.
- Hypersensitivity is due to loss of one copy of the POLR2A gene.
- This lies close to the p53 gene (TP53) on chromosome 17.
- This is often deleted during tumour development and sometimes deletion also removes the adjacent POLR2A gene.
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