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Lecture 1
The lac operon: essential features of a genetic
regulatory circuit for an inductive system
Thursday, 1 November 2012
DNA
RNA
Proteins
Thursday, 1 November 2012
1
2
5
3
4
C
John Gurdon cloning experiments with XenopusGenetic information is not lost during development and can be recalled upon demand.
Thursday, 1 November 2012
DNA
RNA
Proteins
?
?
Thursday, 1 November 2012
Genetics
Biochemistryand Cell Biology
Thursday, 1 November 2012
•The Lac operon decides to use or not to use lactose
•The λ phage deciding whether to sail or to jump in an E. coli cell
•Galactose utilization in yeast
•Making stripes in the Drosophila embryo
Thursday, 1 November 2012
•The Lac operon decides to use or not to use lactose
•The λ phage deciding whether to sail or to jump in an E. coli cell
•Galactose utilization in yeast
•Making stripes in the Drosophila embryo
Thursday, 1 November 2012
Glucose Lactose
Growth
[X]
X=
When presented with a mixture of glucose and lactose, E. coli uses glucose first.
Thursday, 1 November 2012
Glucose Lactose
+
+ +
+
-
- -
-
Not made
not made
Not made
Made
Growth substrate
ß-galactosidase
Thursday, 1 November 2012
Glucose Lactose Mutants
+
+ +
+
-
- -
-
Not made
Not made
Made
Made
Not made
Made
Made
Growth substrate
ß-galactosidase
not made
Thursday, 1 November 2012
ß-galactosidase permease transacetylaseRepressor
Z Y AI
The lac operon
Thursday, 1 November 2012
Repressor
Z Y AI P O
IPTG is an inducer which is not metabolized
Repressor
Allolactose
Lactose
Thursday, 1 November 2012
Glucoseonly
Lactoseonly
ß-galactosidase in
I
I
-
s
OFF ON
ONON
OFF OFF
Mutants
Thursday, 1 November 2012
Z Y AI P Oi -
Repressor mutants
This is a trans recessive mutant: if I is provided from another piece of DNA, it would work
Thursday, 1 November 2012
Z Y AI P Oi -
Repressor mutants
This is a trans recessive mutant: if I is provided from another piece of DNA, it would work
IPTGIPTG
i s
This is a trans dominant mutant: if I is provided from another piece of DNA, it would work
Thursday, 1 November 2012
Repressor
Z Y AI O
P O
!
Thursday, 1 November 2012
Glucoseonly
Lactoseonly
ß-galactosidase in
O c
s
OFF ON
ONON
OFF OFFO
WT
Mutants
Thursday, 1 November 2012
Repressor
Z Y AI P O
Operator mutants
Oc
The repressor cannot bind the operator. This is a cis dominant mutant: it only works on the DNA linked to the mutation.
Thursday, 1 November 2012
Repressor
Z Y AI P O
Operator mutants
Oc
The repressor cannot bind the operator. This is a cis dominant mutant: it only works on the DNA linked to the mutation.
O s
The repressor cannot unbind, even in the presence of IPTG. This is a cis dominant mutant: it only works on the DNA linked to the mutation.
Thursday, 1 November 2012
How to achieve Specificity and Uniqueness
Genome: 4.6 x 10 6
Average protein:DNA contact, 5/6bp
4 6= 5x103
4 11≈ 5x106 ⇒ 11bp binding site
Dimers (2x1) or combinations (1+1)
Repressor
Thursday, 1 November 2012
Z Y AI O
P O
!
Thursday, 1 November 2012
NB: i- [Z(glucose)< Z (lactose)]
Thursday, 1 November 2012
Glucoseonly
Lactoseonly
ß-galactosidase in
CAP
OFF ON
“on”OFF
WT
Mutants
Thursday, 1 November 2012
Glucoseonly
Lactoseonly
ß-galactosidase in
CAP
OFF ON
“on”OFF
WT
Mutants
CAP: Catabolyte activator protein binds cAMP
Glucose cAMP
Glucose cAMP
CAP
CAP active
Thursday, 1 November 2012
σα
CAP + cAMP
Catabolite Activating Protein (CAP) when bound by cAMP forms a dimer and helps RNA polymerase
Thursday, 1 November 2012
σα
CAP + cAMP
Catabolite Activating Protein (CAP) when bound by cAMP forms a dimer and helps RNA polymerase
Mutations in the CAP coding gene result in low expression of ß-galactosidase in the absence of glucose and the presence of lactose
σα
Thursday, 1 November 2012
Glucose Lactose
Growth
[X]
X=
CAPOperator
Lac Promoter
Low High
Occupied Free
Off On
Thursday, 1 November 2012
CAP P O
Z Y A
Thursday, 1 November 2012
Thursday, 1 November 2012
Glucose Lactose
Promoterin
glucose
+
+ +
+
-
- -
-
Lowly
Poised
Loaded
Growth substrate
ß-galactosidase
Not made
“not made”
Not made
Made
CAP
Low
Low to High
High
High
Lowly
Thursday, 1 November 2012
How do repressors and activators actually work?
Thursday, 1 November 2012
There is about four (4) molecules of the Repressor
per cell
Remember Avogadro’s number: one mole contains 6X10 molecules23
Thursday, 1 November 2012
Z Y Alactose
Thursday, 1 November 2012
Z Y Alactose
Thursday, 1 November 2012
Confinement within a cell
Polymerization
Thursday, 1 November 2012
Thursday, 1 November 2012
Cooperativity: when the energetic outcome of two otherwise independent event is thermodynamically more stable than would be predicted from the individual and independent
events
Keq1, 2 is > Keq1 + Keq2
Thursday, 1 November 2012
LacI uses cooperativity to increase functionality
Classic Jacob Monod
Thursday, 1 November 2012
σα
CAP + cAMP
Catabolite Activating Protein (CAP) when bound by cAMP forms a dimer RECRUITS RNA polymerase and USING the weak interacting forces
enhances its functionality
Regulated recruitment; a variation on cooperativity
Thursday, 1 November 2012
Confinement within a cell
Polymerization
Cooperative interactions
Regulated recruitment(d)
Thursday, 1 November 2012
How to achieve Specificity and Uniqueness
Genome: 4.6 x 10 6
Average protein:DNA contact, 5/6bp
4 6= 5x103
4 11≈ 5x106 ⇒ 11bp binding site
Dimers (2x1) or combinations (1+1)
RepressorCAP
Thursday, 1 November 2012
Do bacteria have memories?
Thursday, 1 November 2012
ß-galactosidase
Lactose
Thursday, 1 November 2012
ß-galactosidase
Lactose
Please notice that there is a difference between measuring the ‘average’ amount of ß-gal and the number (fraction) of cells that for a given lactose concentration express ß-gal !!!!!!!!!!!
Thursday, 1 November 2012
memory in bacteria?
Lactose
Thursday, 1 November 2012
Z Y Alactose
There is about 1 molecule of the repressor
Thursday, 1 November 2012
Z Y Alactose
•There is about 1 tetramer of the repressor•The transacetylase (Y) determines the degree of function of I
Z Y A
Thursday, 1 November 2012
Z Y Alactose
low lactose low lactose
or
cell1
low lactose low lactose
cell2
Thursday, 1 November 2012
low lactose low lactose
or
cell1
low lactose low lactose
cell2
Z Y A Z Y A Z Y A
Thursday, 1 November 2012
Z Y A Z Y A Z Y A
low lactose low lactose
or
cell1
low lactose low lactose
cell2
I lacZYA
lactose Y
I lacZYA
lactose Y
Thursday, 1 November 2012
Z Y A Z Y A Z Y A
low lactose low lactose
or
cell1
low lactose low lactose
cell2
I lacZYA
lactose Y
I lacZYA
lactose Y
lac Operon
Thursday, 1 November 2012
That which is found to be true for E coli will be true for the elephant
J. Monod
Thursday, 1 November 2012
Summary•Genetics is a tool that allows us to access mechanisms
•Phenotypic decision making is underpinned by genetic networks
•Genetic networks have a biochemical basis
•Molecular principles of biological processes1. Regulation of local concentration through confinement or polymerization2. Cooperativity3. Regulated recruitment4. Network organization and activity
•In the end all is an evolutionarily selected combination of “Chance and Necessity”
Thursday, 1 November 2012