Genetics, Lecture 12 (LEcture Notes)

8/8/2019 Genetics, Lecture 12 (LEcture Notes)

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110Regulation OfProkaryotic GeneNabeel BasheerMOHAMMAD AL- RUSAN

8/11/2010


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What I want to talk today about it, is the regulation of geneexpression in prokaryotes, We already talked about the generegulation and expression in eukaryotes, talking about thedifferent transcription factors and how they bind to the

promoter, response elements, enhancers, etc. and we talkedabout the leucine zipper and the zinc fingers transcriptionfactors and how their structure regulates transcriptions.What I am going to talk about today is the regulation of gene

expression in prokaryotes and I'll talk briefly about one

system.

What I'm going to talk about today is something called Lac

Operon do you know about it?

Lac means lactose which is a disaccharide carbohydrate

made of glucose and galactose.

Operon means unit of transcription that is composed of

regulatory sequences and structural genes.

So once you have regulatory sequences associated with

structural genes that simply called Operon.

And since we are talking about lactose metabolism or the gene

that responsible about lactose metabolism so the whole set is

called Lac Operon.

the enzyme that hydrolyze lactose is called Beta-

galactosidase (B-galactosidase) and it will be hydrolyzed

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into glucose and galactose. Now we will look at the anatomy of

this Operon.

Lac repressor b-galactosidase

permease acetylase

It has regulatory sequences: Lac I (Lac repressor) , P-O

(promoter-operator), Lac z (responsible for producing or

expressing B-galactosidase) , Lac Y( it's another gene

expressing enzyme called permease. which help to transform

lactose ), Lac A (another structural gene that is called

acetylase: Unknown function).

Promoter binds CAP and RNA polymerase.

CAP abbreviated forcAMP activating protein , or sometimes

it's called (catabolized activating protein) and RNA

polymerase, both of these proteins will bind to the promoter.

And the Lac repressor, protein that is produced from this Lac I

regulatory sequence will bind to the operator.

Operator: is like promoter specific regulatory sequences, this

is specific to bind CAP and RNA polymerase and this is specific

to bind Lac repressor.

Lac repressor: means it will repress the expression ofB-

galactosidase.

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Both promoter and operator have specific regulatory sequence

,that means each has a specificity to their own proteins.

Regulation of the lactose Operon negative control:- Lac repressor is a tetrad (4 subunits) protein it binds to the

operator and once it binds to the operator it blocks the binding

of RNA polymerase, because the operator and the promoter

sequences are overlapped, so once the repressor is bound

RNA polymerase will be unable to bind and as the result therewill be no transcription for these structural genes.

LACTOSE B-galactosidase GLUCOSE + GALACTOSE

So when the microorganism (ex. E.coli) dose not want B-

galactosidase (it just depends on glucose as an energy

source), the repressor binds to the operator and prevent RNA

polymerase to bind, thus no expression for B-galactosidase

in the presence of the repressor.

A molecule called allolactose it's an intermediate metabolite

of lactose metabolism, when you feed microorganism with

lactose, lactose will metabolite to intermediate call

allolactose, and this allolactose is specific to bind to the

repressor, sometimes allolactose is called inducer,there are

other artificial inducers like IPTG (isopropyl thiogalactoside),

and those inducers are positive allosteric effectors ,those

inducers are specific to bind to the repressor and when they

bind ,they will cause a conformational change of the

repressor ,thus the binding affinity for the repressor to the

operator will be low, However the repressor could bind to the

operator in the presence of the inducers (allolactose and

IPTG) .

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We can summarize the action of the inducer of the Lac

Operon in five steps:

1. when lactose becomes available, it is taken up by the

cell.

2. allolactose (an intermediate in the hydrolysis of lactose)

is produced.

3. one molecule of allolactose binds to each of the

repressor subunits.

4. binding of allolactose results in a conformational change

in the repressor.

5. the conformational change results in decreased affinity

of the repressor for the operator and dissociation of the

repressor from the DNA.

Even though we have lactose as a source of carbon it will

bind to the repressor, but still we dont have enough amount

of this inducers (allolactose and IPTG) to dissociates

completely the repressor from the operator.

So under this condition when lactose is available and we have

limited numbers of molecules of inducers like allolactose and

IPTG there will be no transcription for B-galactosidase.

When the repressor is completely bound to the inducer

allolactose ,it will dissociate from the operator , still the RNA

polymerase is unable to bind even the repressor is not

anymore bound so no transcription will take place.

Why is RNA polymerase is unable to bind to the

promoter even the repressor is not bound ??

-it requires transcription factor (activator proteins) ,because

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without these factors the binding affinity for the promoter to

RNA polymerase is very low, so RNA polymerase cannot form a

stable complex with the promoter.

In [slide#6] there is a table shows: Affinity of Lac

repressor for DNA .

the binding of the repressor to Lac operator is 2*10^13 while

for any piece of DNA the binding affinity is 10^6 (very low

binding affinity), but with very high binding affinity to the

operator , because there is a specific binding (as we

mentioned earlier) NOT a random binding.

In the presence ofinducers:

the binding affinity for the repressor to the operator will

decrease.

the binding affinity for the repressor to any piece of DNA

will not change.

the specificity will decrease.

-Specificity is the ratio of:

(Ka for binding to operator DNA) / (Ka for binding

to random DNA)

Regulation of the lactose Operon - positive control:

-To express B-galactosidase we need lactose and glucose.

-in the presence of both lactose and glucose it is not necessary

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for the cell to metabolize lactose for energy because it will

use glucose as the source of energy, it's favorable.

- In the absence of glucose and in the presence of lactose it

becomes advantageous to make use of the available lactosefor energy.

- In the absence of glucose: cells will synthesize cyclic

AMP (cAMP) which is an activator for metabolism, which

serves as a positive regulator of catabolite operons (Lac

Operon),binding ofcAMP to CAP (catabolize activating

protein) increase the affinity for the promoter to CAP

,thus increasing the affinity for the promoter to RNA

polymerase.

- In the presence of glucose and lactose there will not be

enough cAMP to activate CAP, as a result no binding of

RNA polymerase, thus no transcription.

- In the absence of glucose there will be high concentration

Of cAMP it will bind to CAP, at the same time in the

presence of inducer, inducer will bind to the repressorand it will prevent the repressor from binding to the

operator, so RNA polymerase will be available to bind

to the promoter in the presence of activated CAP.

So we have a condition here; the inducer is available that will

prevent the repressor from binding, cAMP is there that will

bind to the CAP, and thus RNApolymerase could bind, andonce RNA polymerase under this condition, LacOperon will

be expressed, so as we see there is an interaction between

(repressor, inducer and metabolite protein) in order to

regulate or control the expression of the specific structural

enzymes I call that the lactose metabolism.

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So we have negative control and positive control in the

presence and absence of inducers and the presence and

absence ofglucose, depends on the physiological condition of

the cell in the presence or the absence of the cAMP, glucose

or lactose, the Lac Operon will be regulated under these

different conditions.

Made by: MOHAMMAD AL-RUSAN

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Genetics, Lecture 12 (LEcture Notes)