MBB331_Week9-Fall-2011

Bait dna this will result in functional transcription

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Unformatted text preview: GIVEN SEQUENCE FACTOR(S) -in reverse expt to ChIP, this approach can find transcription factors binding to given DNA sequence -the DNA sequence of interest (“bait”) is inserted upstream of the minimal promoter of a reporter gene as multiple tandem copies -cDNA library is expressed as fusion to a transcriptional activation domain (AD) -if expressed protein binds to bait DNA, this will result in functional transcription factor and reporter gene expression -one then sequences cDNA to determine identity of binding protein CONTROL OF REGULATORY TRANSCRIPTION FACTORS • Lewin Genes VII 2000 Oxford University Press Activation of inducible Activation transcription factors: (i) (i) tissue specific expression eg. homeoproteins; (ii) eg homeoproteins; (ii) modification controlled - e.g. HSTF phosphorylation leads to activation; (iii) ligand (iii) binding - steroid receptors move from cytoplasm to nucleus; (iv) cleavage - eg. (iv) eg absence of sterol causes cleavage of factor bound to nuclear envelope and ER, releasing it to nucleus where it is active it CONTROL OF REGULATORY TRANSCRIPTION FACTORS • • Lewin Genes VII 2000 Oxford University Press (v) inhibitor release - e.g. NFκ B sequestered in sequestered cytoplasm by I-κ B, in cytoplasm I- B, response to signal, NF-κ B response released from I-κ B and released and moves to nucleus to activate transcription transcription (vi) homo- or heterodimerization -eg. HLH dimerization proteins- binding partners that activate or inactivate a factor factor TRANSCRIPTION FACTOR FAMILIES: Zinc FINGER MOTIFS Zinc FINGER MOTIFS DNA binding β -sheet • Zn-finger: first described in Zn-finger: TFIIIA (Aaron Klug 1985), required by RNAP III for 5S rRNA transcription rRNA • Repeated unit in TFIIIA: 9 X 30-residues always with 2 30-residues cysteines & 2 histidines cysteines • 1 Zn per repeat bound by Cys & Zn His in tetrahedral structure His • Zn fingers usually tandem Zn repeats (but can be more than one group of fingers with ~2-9 fingers in total) fingers Weaver 2002 Molecular Biology Ed. 2 TRANSCRIPTION FACTOR FAMILIES: Zinc FINGER MOTIFS Zinc FINGER MOTIFS -Zinc fingers take their names from the finger like structures that result from binding of zinc ions -the “classic” zinc finger prote...
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