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lecture9 - Eukaryotic RNA polymerases 901 Identifying...

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901 Eukaryotic RNA polymerases
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902 Identifying eukaryotic RNA polymerases using an assay for biochemical activity protein extract source: calf thymus, Xenopus oocytes, Drosophila embryos, Human cancer cells, yeast template: dsDNA (nicked) detection: 32 P-NTP incorporation into insoluble fraction fractionating extract by chromatography
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903 Column Chromatography Fig.5.5 chapter 5.2C ion exchange gel filtration hydrophobic affinity
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904 RNA polymerases: three biochemical activities (R.G. Roeder 1969)
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905 There are three eukaryotic RNA polymerases 1. prepare Nuclear Extract 2. bind Extract to column 3. Fractions are collected as the proteins are eluted from the column 4. Fractions are tested for protein and for RNA synthesis activity Nature. 1969 Oct 18;224(216):234-7. Multiple forms of DNA-dependent RNA polymerase in eukaryotic organisms. Roeder RG, Rutter WJ.
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906 high sensitivity for pol II RNA pol II is responsible for transcribing protein-coding genes test three peaks of RNA polymerase activity with α -amanitin
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907 A general transcription inhibitor
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908 RNA pol I - synthesis of most rRNA synthesis, located in nucleoli (ribosomal gene transcription) RNA pol II - mRNA synthesis nucleoplasm RNA pol III - synthesis of 5s rRNA, tRNAs and some other RNAs.
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lecture9 - Eukaryotic RNA polymerases 901 Identifying...

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