1. 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

2. Regulation of Genes involved in Carbon Source Metabolisms
Glucose
Lactose
Arabinose
Sucrose
Ethanol
Others…
Most bacteria need an organic source of carbon
E. coli can use many different molecules (sugars, amino acids, others…)
Maximum growth rate differs for each carbon source
How rapidly and efficiently carbon source is metabolized
Lag period reflects expression of genes required for metabolism of carbon source

3. Diauxic Growth: bacteria use one carbon source at a time
lactose
Diauxy refers to growth pattern shown above when bacteria are grown in medium containing two carbon sources
Bacteria use up all the glucose (or whatever is most efficient) first
Grow at rate characteristic of glucose
Lag reflects synthesis of genes required for lactose utilization
Culture resumes growth at rate characteristic for lactose
Lactose utilization enzymes are regulated – expressed only when lactose is present
(induction or de-repression)
Lactose utilization enzymes are repressed by glucose, regardless of whether lactose is present
Carbon catabolite repression (“catabolism”)
Genetic analysis: look for mutants that cannot express lactose utilization genes under any condition, or that express lactose utilization genes in the absence of lactose, or that express them in the presence of glucose + lactose
glucose

4. Lactose utilization genes: The Lac Operon
ITPG
-Gratuitous inducer
Not substrate for lacZ
Not substrate for lacA
Levels don’t change when Lac operon is induced

5. Mechanism of Induction (de-repression) by Lactose

6. Mechanism of Catabolite Repression by Glucose

8. Genetic Analysis of Lac Operon Regulation lacZ and lacO mutants
Isolate loss of function mutations:
- lactose non-utilizer mutants
promoter, lacZ, lacY (lacA mutants are utilizers)
- constitutive lac operon expressers
repressor, operator
-Aided by development of IPTG as an inducer and X-gal as an artificial substrate
X-Gal is colorless; betagalactosidase cleaves X-Gal into two products, one of which is blue
Procedure for mutants defective in expression:
Mutagenize, plate on IPTG medium + Xgal, look for whites
For constitutive expressers:
Mutagenize, plate on Xgal (no ITPG, no glucose), look for blues
For mutants defective in carbon catabolite repression:
Mutagenize, plate on IPTG + glucose + Xgal, look for blues
Characterize mutants by making partial diploids (diploid for lac operon only)
dominant vs cis-dominant
cis-acting vs trans-acting elements

9. Genetic Analysis of Lac Operon Regulation lacI mutants

10. Genetic Analysis of Lac Operon Regulation lacI super repressor mutants
lacIs mutants cannot bind lactose
Dominant to lacI+ because lacI acts in trans
lacI-d mutants are dominant negative
-repressor is a tetramer
-I-d proteins can bind to WT proteins
- Tetramers containing I-d subunits are nonfunctional

12. Promoter mutants LacZ Promoter Promoter Mutants:
Random Mutations (what is important?)
Deletions and insertions (presence or absence; spacing of adjacent elements)
Site-directed mutations (is this nucleotide important?)
Use of reporter genes to study promoter and other cis-acting regulatory regions
Use lacZ gene without a promoter
Make mutations to your promoter in vitro
Clone the promoter in front of lacZ
Use X-gal to detect expression of your gene fusion
Promoter
LacZ

14. Z Y Gene
Inducer
#1 Result +
#2 Result

15. 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

16. 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?
Z Y Gene
Inducer
#3 Result
#4 Result

17. Sequence-specific DNA Binding Proteins: example of lacI and CAP sites
DNA binding proteins have a general affinity for DNA and a higher affinity for specific DNA sequences
sequence-non-specific and sequence-specific DNA binding properties
Specific binding sites often show two-fold rotational symmetry
- similar concept to palindromic restriction enzyme recognition sites
- read same sequence on both strands
- indicates DNA binding protein binds as (at least) as a dimer
DNA binding proteins tend to change shape of DNA helix
- effects binding of other proteins (positive or negatively)

18. Characterization of cis-acting sequences by DNA foot printing
Foot printing detects region of DNA that is in close contact with a protein
RNAp on a promoter
lacI on an operator
CAP on a CAP binding site
Clone DNA to be analyzed. Prepare it so that one end is labeled with P32
Purify protein of interest
Let protein bind to DNA (protein/DNA complex)
Treat complex with DNA ribonuclease (DNaseI) – cuts between every accessible nucleotide pair
Conditions set up so that DNaseI cuts each molecule one time on average
Protein on DNA protects some nucleotide pairs from DNaseI
Run the reaction on a DNA sequencing gel next to DNA sequencing reaction of the same fragment
Missing bands represent sites not cut by DNAaseI – sites protected by protein – protein footprint on DNA

19. Regulation of Transcription in Eukaryotes
Cis-acting elements
promoter
enhancers (positive acting)
silencers (negative acting)
trans-acting factors
general (or basal) transcription factors
activators
coactivators (or adaptors)
repressors

20. Eukaryotic promoter elements
Three different polymerases, each with their own promoter sequence
Important sites of the Basal Promoter for RNA Pol II shown above
Polymerase plus general (basal) transcription factors bind to basal promoter
Basal promoter alone can be sufficient for transcription, depending on the promoter
- basal level of expression (a certain amount of mRNA)
Binding can be enhanced or prevented by activators or repressors
- can stimulate or decrease transcription initiation form basal promoter
- increased or decreased amount of mRNA
Amount of mRNA is a property of the rate of transcription initiation
- strong binding of RNA Pol II correlates with RNA Pol II on promoter most of the time, frequent initiation of transcription, lots of mRNA
- weak binding, RNA Pol II less often bound to promoter, ...

21. Role of eukaryotic trans-acting transcription factors
General (basal) factors required for RNA Pol binding (I, II, or III)
- examples:
TATA box binding proteins (TBP)
Transcription factor II H (TFIIH)
- multiple proteins and functions including phosphorylation of RNA Pol II
- promotes elongation
- binding of CAP and 3’ end RNA processing factors
Activators bind to enhancers and promote binding of RNA Pol II
Repressors bind silencers and inhibit binding of RNA Pol II
Co-activators bind to basal factors and to activators/repressors - act as adaptors
Mechanism of activation or repression
- direct contact with other factors to promote or inhibit their function
- hinder the binding of other factors (like lacI and RNA Pol in bacteria)
- modify chromatin to increase or decrease folding (prevent or promote acess of sequence-specific binding proteins to DNA)

22. 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

26. Gal4 = Yeast Transcription Activator
Binds to cis-acting sequences “GAL4 Promoter”
DNA binding domain (DBD)
Activates RNA Pol II transcription
DNA Activation domain
Fuse other Activation Domains to Gal4 DBD and you get a functional activator
Fuse other DBDs to the Gal4 AD and you get a functional activator
2-hybrid strategy developed
One hybrid protein is Gal4DBD::proteinX
Other is Gal4AD::proteinY
If X and Y bind together, that brings AD to where DBD is, activates transcription
Transcription monitored by lacZ reporter

27. 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
Lac and Trp operon examples in text
Sigma factors are bacterial analogy to general transcription factors
One sigma factor is required for efficient binding of RNA Pol to a promoter
Different sigma factors determine which class of promoters are transcribed
Example: heat shock sigma factor and heat shock genes (chaperones)
RNA Pol I, Pol II, Pol III
Pol I has C-terminus that is phosphorylated (Capping and Poly A Tailing)
Coordingate expression of 35S rRNA genes by Pol I
Coordinate expression of tRNA, snRNAs, other structural RNAs by Pol III
Activators or Repressors in eukaryotes often have more than one target gene
Cell-type or Tissue specificity
Developmental stage specificity
Metabolic specificity (heat shock example again)

28. Epigenetic Control: Regulation of gene expression by DNA or chromatin modification
X-chromosome inactivation
Inactive chromatin (heterochromatin)
Inactive X is heavily methylated at C residues
Replication of inactive X chromosome: both DNA sequence AND chromatin structure is replicated
Xist = unstable RNA encoded by inactivation center on X chromosome
Xist becomes stable on one X, binds to DNA, somehow promotes heterochromatin assembly
Xist on other X remains unstable

29. Epigenetic Control: Regulation of gene expression by DNA or chromatin modification
Prader-Willi Syndrome = short stature, poor muscle tone, mild mental retardation, compulsive eating disorder
Do to loss of gene expression in a region at the end of the paternally inherited Ch 15
Maternally inherited Ch 15 region is inactivated by modification in DNA/chromatin structure
Methylation of C residues in CpG dinucleotides
Inactive (heterochromatin-like) chromatin structure
Pattern is established during female gametogenesis
Males have little methylation, active chromatin, active genes
Mechanism for PWS inheritance: above = non-disjucntion in both male and female gamete
Male gamete is nullisomic for Ch 15
Female gamete is disomic for Ch 15 (both inactive)
Next slide shows mutations (usually deletions) of male Ch 15 region
- again, loss of gene expression