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slides-12 - #12 Eukaryotic transcription (II) 1. The...

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#12 Eukaryotic transcription (II) 1. The preinitiation complex 2.TFIID 2. TFIIB 3. TFIIH 4. Transcription initiation and elongation Pages 306-315 not covered
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E. coli RNA polymerase core enzyme can start transcription in vitro. But purified eukaryotic RNA polymerase II core enzyme is incapable of starting transcription in vitro. At least 6-7 so called “general transcription factors” (or TFII factors ) are needed to help RNA polymerase II (TFII A, B, D, E, H, I, J) in the initiation process of all RNAP II-transcribed genes. Binding of TFII factors and RNA polymerase II to the promoter forms the “ pre-initiation complex ” that is required to melt DNA to initiate transcription, which is analogous to the "closed complex" formed by sigma factor, promoter, and RNA polymerase in prokaryotes. Many of TFII factors are multiple subunit complex themselves. AT least one general transcription factors is used in elongation. 1. The preinitiation complex
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It is also called gel mobility shift or simply gel shift assay 1. Radioactively label DNA to be studied 2. Incubate labeled DNA with proteins to be studied 3. Run DNA-protein complex in a native gel (no denature) 4. The more proteins bind to DNA, the slower the complex migrates Using electrophorectic mobility shift assay (EMSA) to study DNA-protein interactions shift
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Experiment : 1. Label a promoter DNA 2. Mix DNA with different TFII factors, with or without RNA Pol II 3. Using gel shift assay to detect DNA-protein complex Results : 1. Four different complexes were found, from DA complex (smallest) to DABPolF (largest in this experiment). 2. TFIID is required for DNA binding and formation of each of the 4 complexes. Using gel shift assay to study formation of preinitiation complex by TFII factors
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TFIID : TFIID is a multiple-subunit complex, it is the most critical TFII factor, it binds to TATA box to start assembly of the preinitiation complex. TFIIA may acts together with TFIID TFIIB: TFIIB is a single peptide factor, it bridges RNA polymerase to TFIID and the promoter TFIIH : TFIIH is also a multiple subunit complex, it has ATPase and helicase activities, it is required for preinitiation complex to melt DNA to initiate transcription. TFIIE may act together with TFIIH. TFIIF
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This note was uploaded on 02/20/2009 for the course LS 3 taught by Professor Lin during the Spring '06 term at UCLA.

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slides-12 - #12 Eukaryotic transcription (II) 1. The...

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