LS3W12-10-Eu-transcript-I

LS3W12-10-Eu-transcript-I - #10 Eukaryotic transcription(I...

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#10 Eukaryotic transcription (I) 1.Eukaryotes RNA polymerases (RNA pol., RNAP) 2. Eukaryotic promoters 3. Enhancers and silencers 4. The preinitiation complex 5. The function of TFII factors: TFIID, TFIIB, and TFIIH 6. Transcription initiation and elongation Pages 252-258, and 299-306 are not covered
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1. Transcription starts at the promoter 2. RNA polymerases have similar structures in prokaryotes and eukaryotes 3. Regulation of transcription initiation is the most common way to control gene expression 4. Transcription is regulated by the DNA regulatory sequences and transcription factors in both prokaryotes and eukaryotes. The principle of transcription regulation is similar in prokaryotes and eukaryotes
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1. Eukaryotic RNA polymerases 1. There are 3 different eukaryotic RNA polymerases (RNA Pol. or RNAP); each transcribes different types of genes 2. The three enzymes in the cell can be separated by chromatography and distinguished by their relative sensitivity to α -amanitin, a cyclic peptide found in some poisonous mushrooms. We will focus on RNA polymerase II, which transcribes all protein-encoding genes. RNA Polymerase - + ++ RNA polymerase I (RNAPI) for most rRNA genes RNA polymerase II (RNAPII) for all mRNA genes RNA polymerase III (RNAPIII) for tRNA, 5S rRNA, etc Sensitivity to α -amanitin In vitro transcription assay
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Due to structural differences, three eukaryotic RNA polymerases have different sensitivity to α -amanitin, which is the toxin found in poisonous mushrooms of Amanita genus, such as “death cap” and “destroying angle”. RNA Pol I is insensitive and RNA Pol II the most sensitive to α -amanitin.
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RNA polymerases have multiple subunits 1. The 3 RNAP have slightly different subunit composition, each has about 11-15 subunits . 2. The major subunits of eukaryotic RNAPs are structurally and functionally similar to that of the prokaryotic RNAP: RPB3 ( α ), RPB2 ( β ), RPB1 ( β '). 3. Some common subunits are shared by all three eukaryotic RNAPs.
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Eukaryotic and prokaryotic RNA polymerases have similar structure
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1. The amino acids Ser , Thr , and Tyr can be phosphorylated by hydrolysis of ATP to add a Pi at the OH group. His can also be phosphorylated, especially in prokaryotes. 2. Protein phosphorylation is catalyzed by protein kinase . Protein phosphorylation is reversible, a phosphorylated protein can be dephosphorylated by a protein phosphatase . 3. Protein phosphorylation often changes protein structure and activity, so reversible phosphorylation is a key regulatory mechanism. Protein modification phosphorylation
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Epitope : the small (~10-20 aa) region of a protein that is recognized by the antibody. Myc, HA, Flag are some commonly used epitopes. Purpose : to rapidly purify a protein complex. Method : 1. Prepare a plasmid expressing a transgene encoding a subunit of a large protein complex fused (in frame) to the sequence encoding an epitopoe for which antibody is available.
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