Transcription Basic Methods

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Presentation transcript:

Transcription Basic Methods Transcript Mapping Upstream and downstream Primer extension assay S1 nuclease assay Promoter Mapping Finding the DNA regions that play a role in regulating a gene’s expression Reporter Genes Transactivation assay DNA-Protein Interactions Cis-Acting Control Elements and Trans-Acting Factors Footprinting Band Shift (Electrophoretic Mobility Shift Assay) DNA-Affinity Purification

Transcript Mapping Primer extension assay S1 nuclease assay 5’ 3’ 3’

Reporter Genes Reporter Gene Promoter Enzyme that does color reaction ß-galactosidase (Xgal; FDG) ß-glucuronidase (X-gluc; MUG) Luciferase(CoA,Luciferin, ATP) Fluorescent Protein GFP dsRED

Mapping Promoters +++ ++ - + +/- Reporter Activity

Transactivation Assay Effector Plasmid Driver - Promoter CDS of Transcription Factor CDS of Reporter Protein Test Promoter ? Reporter Plasmid

Transcription DNA-Protein Interactions Promoters and Enhancers Cis-Acting Control Elements Trans-Acting Factors DNA-Protein Interactions Footprinting Band Shift (Electrophoretic Mobility Shift Assay) DNA -affinity Chromatography

DNA-Protein Interactions Footprinting

DNA-Protein Interactions Band Shift Assay

DNA-Protein Interactions DNA-affinity Chromatography

Regulation of Transcription Bacteria: The Lac Operon (Part I)

Regulation of Transcription Bacteria: The Lac Operon (Part II)

Regulation of Transcription Bacteria: The Trp Operon

Regulation of Transcription Eukaryotes: The simple promoter GC-Boxes TATA-Box HSVtk gene CAAT box

Regulation of Transcription Eukaryotes: Enhancers Orientation independent Distance independent Location independent

Regulation of Transcription Eukaryotes: Specific vs Regulation of Transcription Eukaryotes: Specific vs. Basal Transcription Factors

Regulation of Transcription Eukaryotes: Specific Transcription Factors Repressor Activator

Mapping Transcription Factor Domains Effector Plasmid Activity Reporter Plasmid CDS of Transcription Factor Driver - Promoter +++ +++ - +++ - +++

Regulation of Transcription Eukaryotes: Conserved DNA Binding Domains

Regulation of Transcription Eukaryotes: Tissue Specific Regulation Brain Enhancer Liver Enhancer Embryonic Limb Bud Enhancer

Regulation of Transcription Eukaryotes: Multiple Functional Elements

Regulation of Transcription Eukaryotes: Regulation at the Level of Chromatin Acetylation Remodeling

RNA Processing Prokaryotes rRNAs tRNAs Eukaryotes mRNAs

Specialized Processing Systems tRNAs rRNA

Eukaryotic mRNA Processing Overview

Eukaryotic mRNA Processing Polyadenylation

Eukaryotic mRNA Processing Splicing: Methods

Eukaryotic mRNA Processing Splicing: Two Step Reaction

Eukaryotic mRNA Processing Splicing: Spliceosome snRNAs 50-200 nt U1,U2,U5,U6, snRNPs snRNA +6-10 proteins

Eukaryotic mRNA Processing Splicing: Alternative Splicing

RNA Degradation Half life of mRNAs rRNAs and tRNAs: very long mRNAs Bacteria : approx. 2-3 minutes Mammals: < 30 min to >20 hours

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