Basal Transcription

• 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.

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.

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.

• 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.

• 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.

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.

• 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. 

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. 

• 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.

• 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.

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

• 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.

• 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.