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Slides-10 - #10 Prokaryotic transcription(III 1.Transcription switch by changing sigma factors 2.Transcription switch by changing RNA polymerase

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#10 Prokaryotic transcription (III) 1.Transcription switch by changing sigma factors 2.Transcription switch by changing RNA polymerase 3.DNA-protein interactions 4.Long distance DNA-protein interaction Chapter 8, section 8.3 not covered Chapter 9, section 9.1 not covered
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1. Bacteria usually have one major sigma factor to transcribe most genes, plus several minor sigma factors used to transcribe specific group of genes under specific conditions. 2. The major sigma factor in E. coli is σ 70 ( σ A ) , which binds to the TATA box and transcribes most E. coli genes. 3. Minor σ factors recognize promoters with special -10 box different from the TATA box. For example: σ 32 ( σ H ) - respond to heat shock (-10 box: CCCCATNT) σ 54 ( σ N ) - respond to nitrogen supply (-10 box: TTGCA) 4. Although different sigma factors bind to different promoters, they interact with the same core enzyme. 1. Transcription switch by changing sigma factors
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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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e.g.2. A gene with two promoters Some genes may be needed under more than one conditions, so those genes may be transcribed by RNA polymerase with different sigma factors. Because different sigma factors bind to different promoter, such a gene may have more than one promoter, each with its own -35 and -10 boxes. Different promoters of the same gene may overlap in sequence, each with its own transcription start site. e.g. Bacterium Basillus's gene spoVG has two alternative promoters
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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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e.g.3. Bacterial phage may bring its own σ When phage SPO1 infects bacterium Basillus subtilis, it first transcribes so-called " early genes ", using the host sigma factor. One of the early genes encodes a
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This note was uploaded on 02/20/2009 for the course LS 3 taught by Professor Lin during the Spring '06 term at UCLA.

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Slides-10 - #10 Prokaryotic transcription(III 1.Transcription switch by changing sigma factors 2.Transcription switch by changing RNA polymerase

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