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MCB 317 Genetics and Genomics MCB 317 Topic 10, part 2, A Story of Transcription Deletion and Linker Scanner Analysis In vitro Txn Assay Promoter not sufficient in vivo Identify and define TBP and basal factors Extract + Prom.-Enh. Basal Facts. + Prom.-Enh. Activated Txn (Enhanced) & Regulated Txn Extract + Prom.-Enh. Identification of Enhancers Activators Co-activators + Enhancer & TBP & TAFs Promoter “Activated” txn & Regulated txn Identifying Proteins that Regulate Transcription Experimental Continuum • Biochemistry – – – – – Chemistry Enzymology Crystallography Protein purification Molecular biology Proteomics • Genetics – – – – – Population Mutant screens Classical e.g. Epistasis Molecular genetics Molecular biology Genomics Fields-of-study function in conjunction few biologists/biochemists adhere to a particular sub-field “Factors” are required for specific txn of genes in vitro gene gene + RNAPI, II or III + Radioactive nts “Random” initiation + RNAP + radiact. nts + Nuclear Extract Initiation at start Sites and txn of specific genes Conclusions 1. Cellular (nuclear) molecules other than the RNAPs mediate specificity of txn. Different than prokaryotes. 2. “Factors” recruit RNAPs to their Promoters! How to Identify Txn “Factors”? 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 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) Deletion and Linker Scanner Analysis In vitro Txn Assay Promoter sufficient in vitro Identify and define TBP and basal factors Extract + Prom.-Enh. Basal Facts. + Prom.-Enh. Activated Txn (Enhanced) & Regulated Txn Extract + Prom.-Enh. In vivo Txn Assay Promoter not Sufficient Identification of Enhancers Activators Co-activators + Enhancer & TBP & TAFs Promoter “Activated” txn & Regulated txn 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] Order of Addition Assay How to identify where factors bind to genes? 1. Footprinting 2. Gel mobility shift assay (band shift assay) Nucleases • • • • Site specific (restriction enzymes) Exonucleases DNAases RNAases “Footprinting” DNAase I Footprinting DNAase “nicks” one strand Gel Mobility Shift = Band Shift = EMSA (Electrophoretic Mobility Shift Assay) “Native gels = Gels in which proteins remain functional 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 Bandshift = simple assay to monitor purification Assay fractions for factors by band shift gel Gel Mobility Shift Assay = Band Shift Assay “Free” DNA = unbound DNA; only small % needs to be bound Why is small amount of DNA shifted experimentally significant? How does this compare to footprinting? Combine gel shift and Footprinting DNAase -> Run Gel -> cut out shifted bands -> denature -> sequencing gel Lodish 11-13 Affinity purification of DNA binding proteins Which txn proteins bind DNA? • General txn factors? • Activators? “Real” Data: actual basal factors, not hypothetical fxns Therefore, TFIID must contain additional components that are required for activated levels of txn TAFs = TBP Associated Factors 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 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 Stable pre-initiation Complex (PIC) TFIIH TFIIH = Helicase and RNAPII CTD kinase How would you generate the data for a step-wise assembly model? Title “Complex” binds multiple sites Cooperativity: strong sites help binding at weak sites Order of binding- is TBP/TFIID always first? How are txn by RNAP I, RNAP II and RNAP III similar and dissimilar? RNAP I Txn RNAP III Txn Deletion and Linker Scanner Analysis In vitro Txn Assay Promoter sufficient in vitro Identify and define TBP and basal factors Extract + Prom.-Enh. Basal Facts. + Prom.-Enh. Activated Txn (Enhanced) & Regulated Txn Extract + Prom.-Enh. In vivo Txn Assay Promoter not Sufficient Identification of Enhancers Activators Co-activators + Enhancer & TBP & TAFs Promoter “Activated” txn & Regulated txn