lecture_20

lecture_20 - Lecture19cont. Howarepromotersdefined 1. 2. 3....

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Lecture 19 cont.
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How are promoters defined 1. Sequence 2. Activity 3. Direct identification of the 5’ end . 4. Physical methods
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Sequence Gazing Sequence comparison, genomic approaches.
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Activity Fragments of DNA that are capable of expressing a reporter gene to the same level and under the same regulatory conditions. i.e. Promoter cloning.
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Direct identification of the 5’ end The actual 5’ end of the message is unique, compared to a processed 5’ end. The first nucleotide contains a 5’ triphosphate, processed ends would leave a 5’ monophosphate. This 5’ triphosphate is a substrate for the eukaryotic “capping” enzymes, which modifies the 5’ end by adding a 7methyl G by a 5’ 5’ linkage.
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5’ cap
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Reaction 1. The starting point is the unaltered 5' end of an RNA molecule. This features a final nucleotide followed by three phosphate groups attached to the 5' carbon. 2. One of the terminal phosphate groups is removed (by a phosphatase), leaving two terminal phosphates. 3. GTP is added to the terminal phosphates (by a guanylyl transferase), losing two phosphate groups (from the GTP) in the process. This results in the 5' to 5' triphosphate linkage. 4. The 7 Nitrogen of guanine is methylated (by a methyl transferase). 5. Other methyltransferases are optionally used to carry out methylation of 5' proximal nucleotides.
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Physical methods: Such as DNA foot printing One actually looks at where the Polymerase and Promoter interact. Look at protection of the DNA from DNAses or chemical cleaving reagents, (such as OH . ) where DNA is bound to protein.
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Promoters Promoter consensus sequences: σ 70 TTGACA. ....... 16 17. ........... TATAAT How was this consensus derived? 10 35
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Promoter comparisons Probability of that particular base being present in that position was compiled using several hundred promoters. Spacing T T G A C A T A T A A T 43 69,79,61,56,54,54 77,76,60,61,56,82
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Promoter mutations, making specific changes at each position and examining the effect on transcription. For example: A mutation in the 33 G to an A decreases promoter activity by about 8 fold, at the P ant promoter.
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In addition to these “core” sequences, additional cis sites and proteins are used to modulate transcription. In particular, highly expressed promoters usually have additional sites and interactions aiding in the process
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Regulation I, at the promoter 1) Regulate intrinsic strength by sequence. Closer to consensus, the stronger the promoter 2) Regulate by which sigma drives the promoter. Want the promoter on during a heat shock, use σ 28 , during Nitrogen starvation, σ 54 , etc.
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Regulation II, turning it up or down Regulon: a series of genes that are coordinately regulated and in general are all involved in a related activity these include: Heat Shock, nitrogen starvation, carbon utilization, anaerobiosis, etc.
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4 general regulatory systems Simple systems : metabolites acting as co repressors and/or inducers Global Control
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This note was uploaded on 12/02/2009 for the course MIC 140 taught by Professor Meeks,singer during the Spring '09 term at UC Davis.

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lecture_20 - Lecture19cont. Howarepromotersdefined 1. 2. 3....

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