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MCB 317 Genetics and Genomics

MCB 317 Genetics and Genomics. MCB 317 Topic 10, part 2, A Story of Transcription. Deletion and Linker Scanner Analysis. Identify and define TBP and basal factors. In vitro Txn Assay. Extract + Prom.- Enh. Basal Facts. + Prom.- Enh. Promoter not sufficient in vivo. Activated Txn

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MCB 317 Genetics and Genomics

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  1. MCB 317Genetics and Genomics MCB 317 Topic 10, part 2, A Story of Transcription

  2. Deletion and Linker Scanner Analysis Identify and define TBP and basal factors In vitro Txn Assay Extract + Prom.-Enh. Basal Facts. + Prom.-Enh. Promoter not sufficient in vivo Activated Txn (Enhanced) & Regulated Txn Extract + Prom.-Enh. Identification of Enhancers Activators Co-activators + Enhancer & TBP & TAFs Promoter “Activated”txn & Regulated txn

  3. Identifying Proteins that Regulate Transcription

  4. Experimental Continuum • Biochemistry • Chemistry • Enzymology • Crystallography • Protein purification • Molecular biology • Genetics • Population • Mutant screens • Classical e.g. Epistasis • Molecular genetics • Molecular biology Proteomics Genomics Fields-of-study function in conjunction few biologists/biochemists adhere to a particular sub-field

  5. “Factors” are required for specific txn of genes in vitro gene gene + + RNAP RNAPI, II or III + radiact. nts + Radioactive nts + Nuclear Extract Initiation at start Sites and txn of specific genes “Random” initiation

  6. Conclusions Cellular (nuclear) molecules other than the RNAPs mediate specificity of txn. Different than prokaryotes. 2. “Factors” recruit RNAPs to their Promoters!

  7. How to Identify Txn “Factors”?

  8. Controls allow interpretation of results. One way to look at controls: Positive control insures that Assay or experiment is working properly. Negative control sets the “baseline” or defines the “signal-to-noise” ratio Controls need to be included in virtually all experiments

  9. RNAPII Txn • General transcription factors: TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH • General factors also called Basal Factors • Basal factors plus RNAPII sufficient for txn in vitro from a gene and promoter • Required for txn of all RNAPII genes • Factors are (protein) complexes • Subunits: TFIIA = 2, TFIID ~ 15, TFIIE = 2, TFIIF = 3, TFIIH = 10 subunits • “Core” RNAPII = 12 subunits in yeast • Factors v. Fractions (Fxn)

  10. Deletion and Linker Scanner Analysis Identify and define TBP and basal factors In vitro Txn Assay Extract + Prom.-Enh. Basal Facts. + Prom.-Enh. Promoter sufficient in vitro Activated Txn (Enhanced) & Regulated Txn Extract + Prom.-Enh. In vivo Txn Assay Promoter not Sufficient Activators Co-activators + Enhancer & TBP & TAFs Promoter “Activated”txn & Regulated txn Identification of Enhancers

  11. What do factors do? • Do they bind DNA? • Are they enzymes that modify the polymerase? • Do they bind RNAPII (ala Sigma Factor)? • Do they do any of a hundred other things? • Are they artifacts? [TFIIC, TFIIG]

  12. Order of Addition Assay

  13. How to identify where factors bind to genes? Footprinting Gel mobility shift assay (band shift assay)

  14. Nucleases • Site specific (restriction enzymes) • Exonucleases • DNAases • RNAases

  15. “Footprinting” DNAase I Footprinting DNAase“nicks” one strand

  16. Gel Mobility Shift = Band Shift= EMSA(Electrophoretic Mobility Shift Assay)

  17. “Native gels = Gels in which proteins remain functional

  18. How to purify factors that bind DNA?How to assay for DNA binding proteins? Footprinting Gel mobility shift assay = Band shift assay Affinity purification

  19. Bandshift = simple assay to monitor purification

  20. Assay fractions for factors by band shift gel

  21. Gel Mobility Shift Assay = Band Shift Assay “Free” DNA = unbound DNA; only small % needs to be bound

  22. Why is small amount of DNA shifted experimentally significant? How does this compare to footprinting?

  23. Combine gel shift and Footprinting DNAase -> Run Gel -> cut out shifted bands -> denature -> sequencing gel

  24. Lodish 11-13

  25. Affinity purification of DNA binding proteins

  26. Which txn proteins bind DNA? • General txn factors? • Activators?

  27. “Real” Data: actual basal factors, not hypothetical fxns

  28. Therefore, TFIID must contain additional components that are required for activated levels of txn TAFs = TBP Associated Factors

  29. Results without Experiment TFIID is a complex of ~15 proteins: TBP plus 14 TAFs TBP is at the heart of RNAPII Promoter function: it binds TATA it binds TAFs it binds basal factors

  30. A current model for assembly of the general txn factors at promoters Does this process actually occur in vivo? Remember, we’ll talk about it later

  31. Stable pre-initiation Complex (PIC) TFIIH TFIIH = Helicase and RNAPII CTD kinase How would you generate the data for a step-wise assembly model?

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