lec 8 - Activators and repressors of transcription...

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Activators and repressors of transcription • Transcriptional control elements like enhancers are binding sites for regulatory proteins. • These proteins can be identified by biochemical techniques. – DNase I footprinting – Electrophoretic mobility shift assay (also called EMSA or „gel shift‟).
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Experimental Methods • DNase I footprinting experiments reveal specific binding sites for DNA binding proteins. • EMSA or gel shift assays can be used to detect DNA binding proteins during biochemical purification. • Co-transfection assays in cultured cells are used to evaluate whether a protein encoded by a known gene is a transcription factor.
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DNase I footprinting: Logic: If a protein is bound to a specific DNA sequence, it will protect that sequence from nuclease digestion. DNA is end-labelled and partially digested with a nuclease that cuts randomly. Is used to map precise binding sites of a protein to DNA.
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M, size markers; NE, no protein added; O, total protein extract; FT, flow-through, 1-22, different fractions that are released from a column under different salt conditions. binding site Fractions 9-12 contain the DNA binding protein
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EMSA (a.k.a. gel-shift or band-shift assay) • Is better than footprinting for quantitative analysis of DNA-binding proteins. • Doesn‟t provide the specific DNA- binding sequence. • Logic: A segment of DNA bound to a protein will migrate slower in a gel than the DNA alone.
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Fractions 7 and 8 contain the DNA binding protein. The DNA is radiolabelled and contains a known regulatory element.
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"footprint" on a DNA molecule is indicative of a. a lack of interaction between the specific protein and DNA b. protection from DNAse by the specific protein
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lec 8 - Activators and repressors of transcription...

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