lecture15

lecture15 - Eukaryotes regulatory elements potential...

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1601 Eukaryotes regulatory elements potential mechanisms of regulation some experimental techniques
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1602 Lodish Fig. 11-12 Control elements that regulate eukaryotic genes promoter - close to transcription initiation site position/orientation-dependent enhancer - collection of binding sites independent of position/orientation
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1603 How do TFs modulate transcription? - chromatin reorganization to regulate accessibility - PIC assembly: recruiting PIC components - initiation by RNA pol: open complex formation - elongation/ pausing / anti-termination - coordinate splicing, mRNA maturation, export
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1604 How do TFs modulate transcription? Lodish Fig. 11-1
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1605 RNA pol II and Mediator
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1606 Experimental Techniques to study gene regulation In vitro assays Run-off txn assay EMSA DNAse1 footprinting Endogenous genes Northern blot / qPCR Restriction enzyme accessibility assay Chromatin Immunoprecipitation use knockouts / inhibitors Transient transfection assay identify promoter elements by mutational analysis characterize TFs by mutational analysis
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1607 Lodish Fig. 11-16 Transfection assay to characterize TFs and promoter elements Two plasmids: - Reporter plasmid with DNA binding site within promoter driving expression of a reporter gene - Plasmid expressing protein of interest Investigate promoter elements or TF structure function relationship What is a reporter gene?
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lecture15 - Eukaryotes regulatory elements potential...

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