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review-1 - Review of lectures #10-12 Two studies of the...

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Review of lectures #10-12
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Two studies of the function of sigma factor (#10)
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e.g.1. Bacterium Basillus changes its sigma factor for sporulation Sporulating cell vegetative cell Basillus normally grow as vegetative cells ( σ A ), but when conditions get tough, it switches to sporulation by changing gene expression ( σ E ) Experiment 1. Clone a Basillus genomic DNA fragment containing two types (vegetative and sporulating) of genes 2. Using the cloned DNA to run in vitro transcription with 32P-labeled NTP, using core enzyme plus either σ A or σ E . 3. Using labeled RNA product as probe to hybridize a Southern blot of the restriction digested DNA template. Result : the second region is transcribed only by σ E (+core) σ A σ E
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Restriction digest in the middle of the gene A “run off” experiment (p111): 1. Clone the spoVG gene involved in sporulation, and cut in the middle of the gene 2. Purify sigma factors σ B and σ E 3. Using the restriction digested spoVG DNA as the template in an in vitro transcription reaction, using 32 P-labeled NTP, core enzyme, σ B (in lane 1), or σ E (in lane 2), or both (lane 3) 3. Run labeled RNA product in a gel Result : σ B transcribes only from promoter P1 σ E transcribes only promoter P2 σ B and σ E together transcribe both P1 and P2
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Using Trp repressor to show how trp-binding change TrpR protein conformation and its DNA-binding (#10)
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Trp repressor changes conformation when binding to tryptophan For example, insertion of tryptophan to the Trp repressor changes the position of Arg 54, pushing the DNA-engaging helix toward a better fit to the DNA major groove
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Change of conformation of trp repressor allows it to align the position of helix E to better fit the major groove of DNA (-trp) (+trp)
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Why transcription factors often bind DNA as dimers (#10)?
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Advantages of binding DNA as dimers 1. Increased local concentration of DNA-binding motifs 2. Decreases energy needed for binding DNA 3. Increased diversity : one transcription factor protein often dimerizes with not only an identical protein (homodimer), such as lac and trp repressors, but also with different (but same type, such
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review-1 - Review of lectures #10-12 Two studies of the...

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