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

Eukaryotic Transcription How We “Know” What we Know Abbreviation for Transcription = Txn

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

What is “True” Will Change as We Go Through the Story of Txn Our “Knowledge” of Subjects Undergoing Active Research Evolves “Knowledge” -> A Series of Models

Discovery and Identification of Eukaryotic Promoters

Identification of DNA Sequence Elements- General Strategy 1.Quick, rough look- 100’s bp to 10Kb -> example: Reporter Assay 2. Narrow to specific region- 100’s bp -> example: Gel mobility shift 3. High resolution analysis- Identify specific sequence element bps -> example: footprinting, site directed mutagenesis

PCR-based construction of deletion mutants Primer “tail” = BamHI site Primer “tail” = HindIII site PCR Cut with BamHI and HindIII and clone

Deletion Analysis BamHI XhoI HindIII PCR HindIII

Deletion Analysis BamHI XhoI HindIII PCR HindIII BamHI XhoI HindIII PCR HindIII

Deletion Series from the 3’ end BamHI HindIII XhoI BamHI HindIII XhoI BamHI HindIII XhoI BamHI HindIII XhoI

Deletion Analysis Defines the Borders of Control Regions Txn Yes No Something between -80 and -90 nts required for txn

Deletion Analysis Defines the Borders of Control Regions Txn Yes No Something between -80 and -90 nts required for txn Something between +20 and +30 nts required for txn Control Region Between -90 and +30, but how much reqiuired?

Construction of Linker-Scanner Mutant BamHI XhoI HindIII PCR HindIII BamHI HindIII PCR BamHI XhoI BamHI XhoIHindIII

Construction of Linker-Scanner Mutant BamHI XhoI HindIII Linker-scanner mutations are substitution mutations Length of mutant = same length as original clone Wild-type except at the XhoI substitution site

ATGCGATGCTAGCTATTTAGATCGGATCGAATCGATCGATCG ATAGGTC ATGCGATCTCGAG CTCGAGTATTTAGATCGGATCGAATCGATCGATCGATAGGTC ATGCGATCTCGAGTATTTAGATCGGATCGAATCGATCGATCG ATAGGTC ATGCGATGCTAGCTATTTAGATCGGATCGAATCGATCGATCG ATAGGTC ATGCGATCTCGAGTATTTAGATCGGATCGAATCGATCGATCG ATAGGTC

Site-directed Mutagenesis

Use of Oligos to Synthesize Mutant Alleles XhoI “Gap” Txn YES NO

Use of Oligos to Synthesize Mutant Alleles XhoI HindIII BamHI XhoI BamHI HindIII “Gap” Wild-type Synthesized Mutant allele CTCGAGTAGCCGTAGCTCGACTCGAG GAGCTCATCGGCATCGAGCTGAGCTC TAGCCGTGGCTC GA ATCGGCACCGAG CT

Site directed mutagenesis, part 2

Site directed mutagenesis, part 3

Site directed mutagenesis, part 4

Site directed mutagenesis, part 5

Site directed mutagenesis, summary

Mutational/Genetic Analysis of DNA Can be used to Study: Promoters Enhancers Origins of Replication Centromeres Telomeres ORFs any DNA Sequence-dependent Process

Initial Result = Promoters are Sufficient for Txn “Run-off expt.”

Watson 9-5 Several small elements None essential (in this case) Linker-scanner Analysis -> Several Elements

Eukaryotic Promoter Elements -Promoter Elements Conserved Among Eukaryotes -No Individual Element found at All Promoters

Deletion and Linker Scanner Analysis In vitro Txn Assay Define Promoters Promoters sufficient for Txn

Do Promoter Elements function in vivo similarly to the way the function in vitro?

Watson 12-7 Transfection and Electroporation Transient Transfection Assay

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

Ab Protein Expression Pattern Gene Gene (Organism 2) Mutant Gene Biochemistry Genetics Mutant Organism

Molecular Genetics Summary 1.Column Chromatograpy (ion exch, gel filtr) 2.A. Make Polyclonal Ab; B. Make Monoclonal Ab 3.Western blot, in situ immuno-fluorescence (subcellular, tissue) 4.Screen expression library (with an Ab) 5.Screen library with degenerate probe, mass spec. & database 6.Protein expression (E. coli) 7.A. Differential hybridization 8.A. Northern blot, in situ hybridization, GFP fusion, RT-PCR and q-RT PCR 9.A. low stringency hybridization; B. computer search/clone by phone; C. computer search PCR 10.Clone by complementation (yeast, E. coli) 11.A. Genetic screen; B. genetic selection 12.RNAi

Reverse Transcriptase PCR or RT-PCR A Qualitative Test for Whether an mRNA is present

Quantitative PCR or qPCR or Real Time PCR qPCR machine is a PCR machine that can measure the fluorescence of the reaction after each cycle SYBR green

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56 Linear range = cycles 16-24

57 Linear range = cycles 16-24

58 SERIES OF 10-FOLD DILUTIONS

qPCR Can quantify the level of a given RNA in a sample by measuring the number of cycles it takes to produce a “threshold” level of PCR product. The threshold level is the Ct value; which is a value in the linear range of amplification on a logarithmic plot.

qRT-PCR RT-PCR -> qPCR Best method for quantitating levels of an mRNA in a sample

RT-PCR qPCR qRT-PCR

Properties of Enhancers

Enhancers= short regions (typically ~ 200 bp) of densely packed consensus elements Some elements found in both promoters and enhancers Enhancers= different combinations of elements found in other enhancers

Watson 9-5 Several small elements None essential (in this case)

Which element(s) are required for regulated txn? Regulatory Elements v. Control Elements

E1E2PrCoding Region E1E2Pr E1E2 E1Pr Gluc Transcription MetalNeither E2Pr Genes can have Multiple Enhancers Which Regulate Different Responses