MCB 121 Lecture 9

MCB 121 Lecture 9 - MCB 121 - Lecture 9 I. Promoter...

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MCB 121 - Lecture 9 I. Promoter elements II. Biochemical factor identification III. PIC assembly Pol II requires multiple additional factor to transcribe even naked DNA * Pol I and Pol II need the factors as well but not as many as Pol II does. - the transcription factors need to associate with DNA (directly or indirectly) - some transcription factors bind to DNA only at specific site of promoter Methods for Identification of Promoter Elements 1) Mutagenesis and Introduction - take a promoter region (region that we believe it regulates the expression of the gene) - change it and put it back into cell. * one of the gene used: Report gene ( just coding sequence) - hook the promoter up to a coding region for the protein that's easy to assay . - such as CAT, GFP => measure by how much reporter gene activity there is transcription level -120 +1 Location of mutation (transcription of nucleotide relative to the start of transcription) => do the mutation in promoter => at transcription level: 1 is the unaltered promoter level. => look what level of transcription when there is mutation at 120, 60, or -5, etc. => this means the mutation at -80 location and -25 location = huge effect on promoter => this identify that region at -25, -80, and -100 are important for transcription. * -25 is where TATA box locates. (found in several sequences of promoters)
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* -80 is where CAT box locates. * -100 might be the enhancer region, bind other factors (more general but specific for this gene) * the region that shows higher level of transcription level can be the mutation to the binding site to repressor The definition of promoter in eukaryote: whole region that's necessary for transcription, not just the polymerase binding site in prokaryote. To identify what other components that are necessary for transcription => done by biochemically. => purification is totally useless unless there is an assay. Critical development of the assay: Transcription system in vitro: - make extract of nuclei - take DNA and put it back in => with appropriate promoter - use radioactive nucleotides to see that DNA now can make transcript - has lower level of transcript compared to the one in vivo. - doesn't need enhancer but minimal promoter * minimal promoter: sometimes is CAT box or TATA box (often this one) * this allow transcription in vitro but not in vivo. Basal Apparatus
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This note was uploaded on 05/09/2010 for the course MCB 121 taught by Professor Gasser during the Winter '09 term at UC Davis.

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MCB 121 Lecture 9 - MCB 121 - Lecture 9 I. Promoter...

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