Module 3 Lecture 1 PDF

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Notes from module 3 lecture 1...

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Wednesday, 6 April 2016

Lecture 3.1 Eukaryotic RNA polymerases Exam tip for her lectures: *study off the objectives* Transcription - the process of making an RNA copy of DNA - catalysed by enzyme DNA dependant RNA polymerase (RNAP, RNApol) - looks at template strand adds ribonucleotides to 3’ strand - makes a phosphodiester bond between nucleotides Transcription occurs in multiple places in eukaryotic cells: Eukaryotes more complex cell structure compared to prokaryotes Nucleolus (dark region in nucleus)- dark because of so much transcription happening Multiple types of RNA in eukaryotic cells: Protein coding - mRNA Non-coding RNA (ncRNA) - rRNA - tRNA - snRNA - snoRNA - Regulatory ncRNA RNA polymerase II - contains multiple subunits (protein complex) - Rpb# - Rp = RNA pol. b = II as in RNA pol II # is the number e.g. 1 is the largest subunit and 12 is the smallest subunit. - Rpb1 and Rpb2 are catalytic subunits (are essential) - Rob 3,11,6 are complex subunits (are essential) - Specificity factor not in eukaryotes. The role of this is done by multiple transcription factors - Rpb 4,5,4,8,9,10,12 = in vitro = not essential in vivo = stability, chromatin, mRNA processing Structure of RNA pol II: - DNA enters in the cleft - helicase activity inside the cleft - above the cleft is the clamp: inactive is open, active form is closed (holds on to the DNA, helps stabilise the enzyme and stops it from falling off) - The wall bends the template strand into the active site - pore is where dNTPs enter - active site: template base exposed to dNTPs, when correct dNTP binds, new phosphodiester bond forms. 1

Wednesday, 6 April 2016

- Bridge alternates between being bend shape and straight shape, pushing the new base pair away from the active site.

- DNA/RNA hybrid helix: 8-9 bp after the active site, important for stability. Tiny molecule half of RNA and half DNA

- rudder separates DNA and RNA. DNA gets rewound, RNA goes to exit channel. RNA pol II has proof reading abilities: Mismatch detect: tension in the DNA/RNA hybrid helix. Pol II backtracking: 3’ end of RNA goes into pore, nuclease cleaves transcript removing the mistake (cuts at the active site, 3’ end at the active site), transcription resumes. The RNA pol II C-terminal domain (CTD) - is a hexapeptide repeat - domain of the Rpb1 subunit - tyr(1) - ser(2) - pro(3) - thr(4) - ser(5) - pro(6) - ser(7) - phosphorylation kinase - adds - phosphatases - remove (occurs on ser 2 and 5?) CTD is reversibly phosphorylated during transcription - kinase TF11H phosphorylates ser 5 - kinase P-TEFb phosphorylates ser 2

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