MCB 121 Lecture 10

MCB 121 Lecture 10 - MCB 121 Lecture 10 Outline: I....

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MCB 121 Lecture 10 Outline: I. Transcription cycle II. Enhancers III. Regulatory transcription factors. A) DNA binding domains B) activation domains Transcription Cycle: - it's "cycle" because RNA polymerase transcribe more than one RNA, each transcription requires series of step. 1) Assemble of PIC (basal apparatus) a) TFIID binds TATA b) TFIID recruit TFII B and TFII A (this one can come now or later, doesn't matter) c) TFII F binds with Pol II - TFII F can bind to growing PIC then recruit Pol II or - associate with Pol II first than bind to PIC d) TFIIE binds to the complex (so far) - this allows the recruitment of TFII H (requires TFIIE) e) TFIIH complete the PIC assembly. In the absence of nucleotides, PIC is the stable complex sitting on the DNA Nucleotides are required for the next step of transcription. ATP is also required to synthesize RNA and for other functions. TFIIH with the help of TFIIE: - Helicase activities to melt the promoter: (require ATP) * provides single strand to act as template - CTD-kinase activities (require ATP) * phosphorylates CTD - largest subunit of Pol II * this phosphorylation breaks the link of Pol II with other component of PIC to initiate the transcription. - Transcription initiation Only the phosphorylated Pol II can start transcription. -> Pol II can move on and leave other components of PIC behind (still assembled on promoter) although TFII H has to leave. -> multiple Pol II can transcribe a DNA at the same time. Only the dephosphorylated Pol II can join PIC. Following transcription to termination:
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- CTD phosphatase removes phosphates from CTD of PolII - now the dephosphorylated Pol II can bind to another PIC and transcribe another gene. ** Energy efficiency doesn't seem to be a big deal in higher eukaryote! THERE ARE REQUIRED FOR ALL TYPE II GENES - Not regulatory ------------------------ Individual genes in vivo need to be regulated ! * In vitro transcription: ~100 times lower than in vivo even though it's naked DNA instead of chromatin * "nearly" all genes include additional elements necessary for transcription in vivo. (based on deletion assay or mutagenesis assay) ENHANCER : sequences that increased (supported) transcription - without enhancer, transcription level decreases Minimal promoter: TATA box region and the region surrounding it - used in vitro assay to assemble PIC - minimal promoter + reporter in cell = nothing - minimal promoter + enhancer sequence + reporter in cell = transcription occurs. Enhancer can be backward = still works as well. Enhancer can be in either orientation and either position of the DNA Enhancer doesn't need to be right next to the minimal promoter It only needs to be some place close to the gene. Enhancer sequence is not orientation-independent and relatively-position-independent.
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MCB 121 Lecture 10 - MCB 121 Lecture 10 Outline: I....

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