chromatin lecture1 CW.ppt - Chromatin Structure and transcriptional control Christopher Wynder SCCRI-McMaster University Model Systems Much of the

chromatin lecture1 CW.ppt - Chromatin Structure and...

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Chromatin Structure and transcriptional control Christopher Wynder SCCRI-McMaster University
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Much of the analyses of transcription initiation and elongation involves; Much of the analyses of transcription initiation and elongation involves; -identification of sequence elements -identification of sequence elements -isolation of DNA binding proteins -isolation of DNA binding proteins -assaying transcription activity -assaying transcription activity in vitro in vitro -assaying transcription activation -assaying transcription activation in vivo in vivo (e.g. reporter assays) (e.g. reporter assays) Often this has been done using different forms of naked DNA: Often this has been done using different forms of naked DNA: e.g. 1) oligonucleotides corresponding to DNA binding sites of sequence e.g. 1) oligonucleotides corresponding to DNA binding sites of sequence specific DNA binding proteins specific DNA binding proteins 2) circular plasmids incorporating a reporter gene driven by core 2) circular plasmids incorporating a reporter gene driven by core promoter and enhancer sequence elements. promoter and enhancer sequence elements. In these experimental systems the DNA is essentially devoid of DNA binding In these experimental systems the DNA is essentially devoid of DNA binding proteins other than the sequence specific DNA binding proteins being tested proteins other than the sequence specific DNA binding proteins being tested Model Systems Model Systems
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In Vivo Control of Transcription In Vivo Observations: 1. Transcription factors can’t always bind their target sequence. 2. Transcriptional silencing; no matter how much of the transcription factor is in the cell, the target gene cannot be activated. 3. DNA is locked in a protein matrix. This suggests that in addition to sequence specific transcription factors there is additional control, presumably through the protein matrix that is already bound to the DNA. Problem: In vitro models can’t adequately explain the in vivo complexity of transcriptional control.
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Electron micrograph of eukaryotic Electron micrograph of eukaryotic nuclear DNA isolated at lower than nuclear DNA isolated at lower than physiologic salt concentration (<0.15 physiologic salt concentration (<0.15 M KCl) M KCl) -in low ionic strength solution, the -in low ionic strength solution, the DNA has a more extended DNA has a more extended appearance, like “beads on a string” appearance, like “beads on a string” -each bead is 10 nm in diameter -each bead is 10 nm in diameter -Each bead consists of both DNA and -Each bead consists of both DNA and Protein which together comprise the Protein which together comprise the nucleosome. nucleosome. -the nucleosome represents the -the nucleosome represents the smallest structural unit of a smallest structural unit of a chromosome.
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