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Regulation of Transcription (Ch13)
• Genetic Analysis of Regulation of Transcription
– Identification and characterization of cis-acting and trans-acting regulators
• Detailed analysis of the E. coli lac operon
• General information on eukaryotic cis- and trans-acting elements
• Gal4 illustrates the domain structure of trans-acting regulators
– Gal4 system developed into the famous “two-hybrid assay” for interacting proteins
(end of Chapter 9)
• Paradigms for coordinate control of transcription for multiple
genes
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Operons in prokaryotes
Multiple Sigma factors in prokaryotes
Multiple RNA polymerases in eukaryotes
Multiple genes under control of one trans-acting factor
• Other Examples of transcriptional regulatory mechanisms
– Gene rearrangements
– Epigenetic regulation
Regulation of Genes involved in
Carbon Source Metabolisms
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Glucose
Lactose
Arabinose
Sucrose
Ethanol
Others…
Diauxic Growth: bacteria use one
carbon source at a time
lactose
glucose
Lactose utilization genes: The Lac Operon
Genetic Analysis of Lac Operon Regulation
lacZ and lacO mutants
Genetic Analysis of Lac Operon Regulation
lacI mutants
Genetic Analysis of Lac Operon Regulation
lacI super repressor mutants
Promoter mutants
Z Y Gene
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#1 Result
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#2 Result
Regulation of Transcription (Ch13)
• Genetic Analysis of Regulation of Transcription
– Identification and characterization of cis-acting and trans-acting regulators
• Detailed analysis of the E. coli lac operon
• General information on eukaryotic cis- and trans-acting elements
• Gal4 illustrates the domain structure of trans-acting regulators
– Gal4 system developed into the famous “two-hybrid assay” for interacting proteins
(end of Chapter 9)
• Paradigms for coordinate control of transcription for multiple
genes
–
–
–
–
Operons in prokaryotes
Multiple Sigma factors in prokaryotes
Multiple RNA polymerases in eukaryotes
Multiple genes under control of one trans-acting factor
• Other Examples of transcriptional regulatory mechanisms
– Gene rearrangements
– Epigenetic regulation
Genetic analysis of lac operon
regulation using partial diploids:
1. Draw both operons
2. Note of cis-acting mutations
- what is phenotype for each operon
3. Note trans-acting mutations
- does this change result in (2) above?
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#4 Result
Sequence-specific DNA Binding
Proteins: example of lacI and CAP sites
LacI
LacI
Characterization
of cis-acting
sequences by
DNA foot
printing
Regulation of Transcription in
Eukaryotes
• Cis-acting elements
– promoter
– enhancers (positive acting)
– silencers (negative acting)
• trans-acting factors
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general (or basal) transcription factors
activators
coactivators (or adaptors)
repressors
Eukaryotic promoter elements
Role of eukaryotic trans-acting
transcription factors
Genetic Analysis of Regulation
of Transcription in Eukaryotes
Isolate and analyze mutations in cis and trans-acting element
Use recombinant DNA and transgenic cells/animals
-construct gene fusions in vitro
- transcriptional fusions
- translational fusions
- fusions to LacZ coding sequence
- transform gene fusions into cells/organisms
- determine relative levels of Beta-Galactosidase activity
Paradigms for coordinate control of
transcription for multiple genes
Operons in prokaryotes
Multiple Sigma factors in prokaryotes
Multiple RNA polymerases in eukaryotes
Multiple genes under control of one trans-acting
factor
Epigenetic Control: Regulation of gene
expression by DNA or chromatin modification
Epigenetic Control: Regulation of gene
expression by DNA or chromatin modification