Basal Transcription

RNA Polymerase

  • 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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Transcription Factors

TBP

  • 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.

Promoter Recognition

  • TBP binds the TATA box.
  • TAF1 binds inr.
  • TAF6 binds the DPE.
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PIC Assembly

Pre-Initiation Complex

  • 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.

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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Subunit Evolution

  • 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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Elongation

Process

  • 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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Catalysis Mechanism

  • 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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Pol2 CTD

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.

Phosphorylation

  • 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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Chromatin Immunoprecipitation

  • 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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Western Blot

  • 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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CTD Recruitment

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.

Elongation 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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Termination

  • 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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α-amanitin

  • 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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