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

DNA

RNA

Proteins


1

2

5

3

4

C

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


DNA

RNA

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


Glucose Lactose

Growth

[X]

X=

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


Glucose Lactose

+

+ +

+

-

- -

-

Not made

not made

Not made

Made

Growth substrate

ß-galactosidase


Glucose Lactose Mutants

+

+ +

+

-

- -

-

Not made

Not made

Made

Made

Not made

Made

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

I

I

-

s

OFF ON

ONON

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

i s

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


Repressor

Z Y AI O

P O

!


Glucoseonly

Lactoseonly

ß-galactosidase in

O c

s

OFF ON

ONON

OFF OFFO

WT

Mutants


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.


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.


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

P O

!


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


Glucoseonly

Lactoseonly

ß-galactosidase in

CAP

OFF ON

“on”OFF

WT

Mutants


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


σα

CAP + cAMP

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


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


Glucose Lactose

Growth

[X]

X=

CAPOperator

Lac Promoter

Low High

Occupied Free

Off On


CAP P O

Z Y A


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


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


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


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 A


Z Y Alactose

low lactose low lactose

or

cell1


cell2



or

cell1


cell2

Z Y A Z Y A Z Y A


Z Y A Z Y A Z Y A


or

cell1


cell2

I lacZYA

lactose Y

I lacZYA

lactose Y


Z Y A Z Y A Z Y A


or

cell1


cell2

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