CSB349L6 - CSB349H1 Lecture 6 September 29th 2010...

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CSB349H1 Lecture 6 September 29 th , 2010 Transcription II: Promoters and sequence-specific DNA binding proteins. Last week we discussed the structure of chromatin, what enzymes open the DNA to reveal the promoter region. What are the functional units inside the promoter and the regulatory sequence that controls transcription. There are two important classes of regulatory sequences. 1: core promoter. Not involved in gene regulation (how, where, when), but part of the transcription initiation machine. Let the transcription machinery to know where to start. Works direction dependently. Big, surrounds transcription start site. 2: Proximal promoter. Other regulatory sequence are not found in the promoter but do distinguish regulatory information..not part of all promoters, not every gene needs as proximal promoter. Probably not positioned . A few hundred bp. Thick line: introns and UTR, boxes: exons. Upstream region is the promoter.
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CSB349H1 Lecture 6 September 29 th , 2010 How do we characterize promoters? You have some part of the upstream region of the gene. Take the 5’ region and use restriction enzymes to digest it into a series of fragments which will be ligated into a plasmid that has a reporter gene but no promoter. Then you put the series of plasmids into a model organism (e. coli), for each of these fragments do I see expression of my reporter gene? Most expression in plasmids 1 and 2. Infer that there is a proximal promoter near 1 and 2 that leads to full transcription, missing regultroy regions that lead to full transcription. 5 you’ve chopped it so short that you no longer have the information available. Allows you to define the different regions.
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CSB349H1 Lecture 6 September 29 th , 2010 What exactly are the reg sequences w/in my region? Promoter bashing experiment. Make a series of constructs, scan across the promoter, knocking out a small bit of sequences, internal mutations all along. Smashing pieces of promoter out,to figure out what is important. You’ll get an idea of where the important reg sequences are. The more constructs you make and the smaller pieces you make, the best localization. ******Ask him how this is done again*************** Promoter regulatory sequences. TATA box:
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CSB349H1 Lecture 6 September 29 th , 2010 TATA box described as a consensous sequence. You can have more than one base at that region. How can u do an experiment, make a series of constructs T mutation at each location? Put in reporter complex, no, yes, no, yes, no. A lot of constructs are needed to define which region, because there is this degeneracy. You could test is C, G, or A are acceptable. The number of contructs you have to make really becomes large. Way to understand specificity of promoter like TATA.
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CSB349H1 Lecture 6 September 29 th , 2010 Let nature do the work for you. Go out and read everyone elses papers about TATA boxes. There were 400 TATA boxes that were identified in genes. 352 have an A, 35 have a T, 2 have a G, none have a C.
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