Lecture 1 The lac operon: essential features of a genetic regulatory

Lecture 1

The lac operon: essential features of a genetic

regulatory circuit for an inductive system

Thursday, 1 November 2012

Proteins

John Gurdon cloning experiments with XenopusGenetic information is not lost during development and can be recalled upon demand.

Proteins

Genetics

Biochemistryand Cell Biology

•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

•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

Glucose Lactose

Growth

When presented with a mixture of glucose and lactose, E. coli uses glucose first.

Glucose Lactose

Not made

not made

Not made

Growth substrate

ß-galactosidase

Glucose Lactose Mutants

Not made

Growth substrate

ß-galactosidase

not made

ß-galactosidase permease transacetylaseRepressor

Z Y AI

The lac operon

Repressor

Z Y AI P O

IPTG is an inducer which is not metabolized

Repressor

Allolactose

Lactose

Glucoseonly

Lactoseonly

ß-galactosidase in

OFF ON

OFF OFF

Mutants

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

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

This is a trans dominant mutant: if I is provided from another piece of DNA, it would work

Repressor

Z Y AI O

Glucoseonly

Lactoseonly

ß-galactosidase in

OFF ON

OFF OFFO

Mutants

Repressor

Z Y AI P O

Operator mutants

The repressor cannot bind the operator. This is a cis dominant mutant: it only works on the DNA linked to the mutation.

Repressor

Z Y AI P O

Operator mutants

The repressor cannot bind the operator. This is a cis dominant mutant: it only works on the DNA linked to the mutation.

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.

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

Z Y AI O

NB: i- [Z(glucose)< Z (lactose)]

Glucoseonly

Lactoseonly

ß-galactosidase in

OFF ON

“on”OFF

Mutants

Glucoseonly

Lactoseonly

ß-galactosidase in

OFF ON

“on”OFF

Mutants

CAP: Catabolyte activator protein binds cAMP

Glucose cAMP

CAP active

CAP + cAMP

Catabolite Activating Protein (CAP) when bound by cAMP forms a dimer and helps RNA polymerase

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

Glucose Lactose

Growth

CAPOperator

Lac Promoter

Low High

Occupied Free

Off On

CAP P O

Glucose Lactose

Promoterin

glucose

Poised

Loaded

Growth substrate

ß-galactosidase

Not made

“not made”

Not made

Low to High

How do repressors and activators actually work?

There is about four (4) molecules of the Repressor

per cell

Remember Avogadro’s number: one mole contains 6X10 molecules23

Z Y Alactose

Confinement within a cell

Polymerization

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

LacI uses cooperativity to increase functionality

Classic Jacob Monod

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

Confinement within a cell

Polymerization

Cooperative interactions

Regulated recruitment(d)

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

Do bacteria have memories?

ß-galactosidase

Lactose

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 !!!!!!!!!!!

memory in bacteria?

Lactose

Z Y Alactose

There is about 1 molecule of the repressor

Z Y Alactose

•There is about 1 tetramer of the repressor•The transacetylase (Y) determines the degree of function of I

Z Y Alactose

low lactose low lactose

Z Y A Z Y A Z Y A

I lacZYA

lactose Y

I lacZYA

lactose Y

Z Y A Z Y A Z Y A

I lacZYA

lactose Y

I lacZYA

lactose Y

lac Operon

That which is found to be true for E coli will be true for the elephant

J. Monod

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”

Lecture 1 The lac operon: essential features of a genetic regulatory

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