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Lactose Lactose Glucose + Galactose (Glucose-Galactose) Intracellular Lacy Lacz (Permease) (Β-Galactosidase)

The document discusses gene regulation in bacteria using the lactose (Lac) operon in E. coli as an example. It explains how the Lac operon works, including the roles of the repressor protein LacI and the operator site. It describes different types of regulatory mutants: LacI- mutants that are constitutive; LacOc mutants with mutations in the operator site that are also constitutive; and LacI-d mutants with dominant repressor mutations. Genetic tests including dominance tests and cis-trans tests are used to determine whether mutations are in the repressor protein (LacI) or the operator site and whether their effects are cis- or trans-acting in order to characterize these different types of mutations

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Alolika Dutta Roy
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78 views3 pages

Lactose Lactose Glucose + Galactose (Glucose-Galactose) Intracellular Lacy Lacz (Permease) (Β-Galactosidase)

The document discusses gene regulation in bacteria using the lactose (Lac) operon in E. coli as an example. It explains how the Lac operon works, including the roles of the repressor protein LacI and the operator site. It describes different types of regulatory mutants: LacI- mutants that are constitutive; LacOc mutants with mutations in the operator site that are also constitutive; and LacI-d mutants with dominant repressor mutations. Genetic tests including dominance tests and cis-trans tests are used to determine whether mutations are in the repressor protein (LacI) or the operator site and whether their effects are cis- or trans-acting in order to characterize these different types of mutations

Uploaded by

Alolika Dutta Roy
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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Lecture 16

16

Gene Regulation

We are now going to look at ways that genetics can be used to study gene regulation. The
issue is how cells adjust the expression of genes in response to different environmental
conditions. The principles of gene regulation were first worked out by Jacob and Monod
studying the E. coli genes required for cells to use the sugar lactose as a nutrient.

lactose lactose glucose + galactose


(glucose-galactose) intracellular
LacY LacZ
(permease) (β-galactosidase)

cell mass

log
amount
LacY or LacZ
+ lactose

time

The logic of the Lac operon is that the proteins required to use lactose are only made
when their substrate (lactose) is available. This prevents wasteful expression of
enzymes when their substrates are notavailable.

At first, scientists noted that lactose is both an inducer and substrate for the enzymes
of the Lac operon and they therefore concluded that lactose was somehow acting as a
template for the formation of the enzyme. Then compounds were discovered that could
act as inducers but were not themselves substrates for the Lac enzymes. The classic
IPTG, which is an effective inducer of LacZ
example of such a “gratuitous inducer” is IPTG
expression but isn’t hydrolyzed by ß-galactosidase.
CH3
IPTG = galactose—S—CH
CH3
The existence of compounds such as IPTG shows that recognition of the inducer is a
separate molecular event from lactose breakdown.
The next major finding was the discovery of LacI– mutants. LacI– mutants are
constitutive, meaning that they always express β-galactosidase at high levels regardless
of whether there is an inducer present or not. LacI– mutants have apparently lost a
component of the machinery the cell uses to turn off β-galactosidase expression.

The regulatory system turns out to be quite simple and by isolation of mutants and simple
genetic tests Jacob and Monod were able to figure out the following scheme:

The Lac Operon


repressor
protein β-galactosidase permease
RNA
polymerase

P O
LacI LacZ LacY LacA
promoter operator

+ inducer

The idea is that the inducer has a net positive effect on expression because the inducer
is a negative regulator of the repressor, which is itself a negative regulator of the gene
for β-galactosidase.

We will now consider how regulatory mutants can be analyzed genetically. We will use as
examples different mutations in the Lac system but the genetic tests are very general
and can be applied to most regulatory systems.

Dominance test
β-galactosidase
–IPTG +IPTG Interpretation

I+ Z+ – +
I– Z+ + + I– is constitutive

I– Z+ / F’ I+ Z+ – + I– is recessive

I+ Z– – – Z– is uninducible

I+ Z– / F’ I+ Z+ – + Z– is recessive

I+ Z– / F’ I– Z+ – + I– and Z– mutations complement each other


i.e. the mutations are in different genes.
A second type of constitutive mutant inactivates the operator site and is known as a
LacOc mutation. LacOc mutations are dominant as revealed in tests of the appropriate
merodiploids:

–IPTG +IPTG Interpretation

Oc Z+ + + Oc is constitutive

Oc Z+ / F’ O+ Z+ + + Oc is dominant

You might think that on the basis of a dominance test we could tell whether we have a
LacOc or a LacI– mutation. However, life is not so simple, because it is possible to find
LacI– mutations that are dominant. Such mutations are known as LacI-d. They are
dominant because the repressor protein is a tetramer and LacI-d mutant subunits can
combine with normal subunits and interfere with their function.

–IPTG +IPTG Interpretation

I-d Z+ + + I-d is constitutive

I-d Z+ / F’ I+ Z+ + + I-d is dominant

We will now consider a new genetic test that will let us distinguish LacOc (operator
constitutive) from LacI-d (dominant repressor negative) mutations.

Cis/trans test
–IPTG +IPTG Interpretation

I+ O+ Z+ – +
I-d Z+ / F’ I+ Z– + + I-d is dominant in cis or in trans with Z+;
(cis) Therefore we say it is “trans-acting”.

I-d Z– / F’ I+ Z+ + +
(trans)

Oc Z+ / F’ O+ Z– + + Oc is dominant only in cis with Z+;


(cis) Therefore we say it is “cis-acting”.

Oc Z– / F’ O+ Z+ – +
(trans)

If a mutation is cis-acting we take this as evidence that the mutation affects a site on
DNA like an operator. If a mutation is trans-acting we take this as evidence that the
mutation affects a diffusible gene product such as a repressor.

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