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

2. Deletion and Linker Scanner Analysis In vitro Txn Assay Promoter not sufficient in vivo Identification of Enhancers Identify and define TBP and basal factors Extract + Prom.-Enh. Basal Facts. + Prom.-Enh. Activated Txn (Enhanced) & Regulated Txn Extract + Prom.-Enh. 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 Fields-of-study function in conjunction few biologists/biochemists adhere to a particular sub-field Genomics Proteomics

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

8. Conclusions 1.Cellular (nuclear) molecules other than the RNAPs mediate specificity of txn. Different than prokaryotes. 2. “Factors” recruit RNAPs to their Promoters!

9. How to Identify Txn “Factors”?

11. 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

13. 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)

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

15. 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]

17. Order of Addition Assay

18. How to identify where factors bind to genes? 1. Footprinting 2. Gel mobility shift assay (band shift assay)

19. Nucleases Site specific (restriction enzymes) Exonucleases DNAases RNAases

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

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

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

25. How to purify factors that bind DNA? How to assay for DNA binding proteins? 1. Footprinting 2. Gel mobility shift assay = Band shift assay 3. Affinity purification

27. Bandshift = simple assay to monitor purification

28. Assay fractions for factors by band shift gel

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

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

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

33. Lodish 11-13

34. Affinity purification of DNA binding proteins

38. Which txn proteins bind DNA? General txn factors? Activators?

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

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

47. 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

49. 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

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

51. Title

52. “Complex” binds multiple sites Cooperativity: strong sites help binding at weak sites Order of binding- is TBP/TFIID always first?

53. How are txn by RNAP I, RNAP II and RNAP III similar and dissimilar?

54. RNAP I Txn

55. RNAP III Txn

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