1. 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

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

4. Mapping Promoters
+++ ++ - +
+/-
Reporter
Activity

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

6. 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

7. DNA-Protein Interactions Footprinting

8. DNA-Protein Interactions Band Shift Assay

9. DNA-Protein Interactions DNA-affinity Chromatography

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

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

12. Regulation of Transcription Bacteria: The Trp Operon

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

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

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

16. Regulation of Transcription Eukaryotes: Specific Transcription Factors
Repressor
Activator

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

18. Regulation of Transcription Eukaryotes: Conserved DNA Binding Domains

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

20. Regulation of Transcription Eukaryotes: Multiple Functional Elements

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

22. RNA Processing
Prokaryotes
rRNAs
tRNAs
Eukaryotes
mRNAs

23. Specialized Processing Systems
tRNAs
rRNA

24. Eukaryotic mRNA Processing Overview

25. Eukaryotic mRNA Processing Polyadenylation

26. Eukaryotic mRNA Processing Splicing: Methods

27. Eukaryotic mRNA Processing Splicing: Two Step Reaction

28. Eukaryotic mRNA Processing Splicing: Spliceosome
snRNAs nt
U1,U2,U5,U6,
snRNPs
snRNA proteins

29. Eukaryotic mRNA Processing Splicing: Alternative Splicing

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