©2000 Timothy G. Standish Ecclesiastes 3:1, 17 1To every thing there is a season, and a time to...

©2000 Timothy G. Standish

Ecclesiastes 3:1, 171 To every thing there is a season, and a

time to every purpose under the heaven:17 I said in mine heart, God shall judge the

righteous and the wicked: for there is a time there for every purpose and for every work.


Controlling Controlling Gene Expression:Gene Expression:

BacteriaBacteriaTimothy G. Standish, Ph. D.


All Genes Can’t be Expressed All Genes Can’t be Expressed At The Same TimeAt The Same Time

Some genes are needed for the function of all cells all the time. These continually expressed genes are called constitutive genes.

Other genes are only needed by certain cells or at specific times. The expression of these inducible genes is tightly controlled.

For example, pancreas beta cells make the protein insulin by expressing the insulin gene. Neurons, in comparison, don’t express insulin.


Operons Are Groups Of Genes Operons Are Groups Of Genes Expressed By ProkaryotesExpressed By Prokaryotes

The genes grouped in an operon are all needed to complete a given task

Each operon is controlled by a single control sequence in the DNA

Because the genes are grouped together, they can be transcribed together then translated together


The The LacLac Operon Operon Genes in the lac operon allow E. coli bacteria to metabolize

lactose E. coli is unlikely to encounter lactose, so it would be

wasteful to produce the proteins needed to metabolize it unless necessary

Metabolizing lactose for energy only makes sense when two criteria are met:– Other more readily metabolized sugar (glucose) is unavailable

– Lactose is available


The The LacLac Operon - Parts Operon - Parts The lac operon is made up of a control region and four genes:

1 LacZ - -galactosidase - Enzyme that hydrolyzes the bond between galactose and glucose

2 LacY - Codes for a permease that lets lactose across the cell membrane

3 LacA - Transacetylase - An enzyme whose function in lactose metabolism is uncertain

4 Repressor - A protein that works with the control region to control expression of the operon


The The LacLac Operon - Control Operon - Control The control region is made up of two parts:

1 Promoter – Promoters are specific DNA sequences to which RNA Polymerase

binds so that transcription can occur– The lac operon promoter also has a binding site for a protein called

Catabolite Activator Protein (CAP)

2 Operator – The binding site of the repressor protein– The operator is located downstream (in the 3’ direction) from the

promoter so that if repressor is bound RNA Polymerase can’t transcribe


The The LacLac Operon: Operon:When Glucose Is Present But Not LactoseWhen Glucose Is Present But Not Lactose

Repressor Promoter LacY LacALacZOperatorCAPBinding

RNAPol.

Repressor

Repressor

Repressor mRNA

Hey man, I’m constitutive

Come on, let me through

No wayJose!

CAP


The The LacLac Operon: Operon:When Glucose And Lactose Are PresentWhen Glucose And Lactose Are Present


Repressor

Repressor mRNA


CAP

Lac

Repressor

Repressor

X

RNAPol.

RNAPol.

Great, I can transcribe!

Some transcription occurs, but at a slow rate

This lactose has bent me

out of shape


The The LacLac Operon: Operon:When Lactose Is Present But Not GlucoseWhen Lactose Is Present But Not Glucose


Repressor

Repressor mRNA


CAPcAMP

Lac

Repressor

Repressor

X

This lactose has bent me

out of shape

CAPcAMP

CAPcAMP

Bind to mePolymerase

RNAPol.

RNAPol.

Yipee…!


The The LacLac Operon: Operon:When Neither Lactose Nor Glucose Is PresentWhen Neither Lactose Nor Glucose Is Present


CAPcAMP

CAPcAMP

CAPcAMP

Bind to mePolymerase

RNAPol.

Repressor

Repressor mRNA


Repressor

STOPRight therePolymerase

Alright, I’m off to the races . . .

Come on, let me through!


The The TrpTrp Operon Operon Genes in the trp operon allow E. coli bacteria to make

the amino acid tryptophan Trp operon genes encode enzymes involved in the

biochemical pathway that converts the precursor chorismate to tryptophan.

The trp operon is controlled in two ways:– Using a repressor that works in exactly the opposite way

from the lac operon repressor– Using a special attenuator sequence


The TryptophanThe TryptophanBiochemical PathwayBiochemical Pathway

O

-OOC

OH

HN

HH

-2O3P

OH

HH

CH2O

5-Phosphoribosyl--Pyrophosphate PPi

N-(5’-Phosphoribosyl)-anthranilate

COO-

COO-

H

CH2

C

HO

HO

Chorismate

-OOCOH-2O3PO CH2

NH

CH

C C

H

OH

C

H

OH

Enol-1-o-Carboxyphenylamino-1-deoxyribulose phosphate

NH

-OOC CH2

NH3+

C

H

Tryptophan

H2OSerine

Anthranilate

COO-

NH2

Glutamate +Pyruvate

Glutamine

CO2+H2O -2O3PO CH2

CH

C C

H

OH

C

H

OH

NH

Indole-3-glycerol phosphateGlyceraldehyde-3-phosphate

NH

Indole

Anthranilate synthetase(trpE and D)

Anthranilate synthetase

N-(5’-Phosphoribosyl)-anthranilateisomerase Indole-3’-glycerol phosphate synthetase (trpC)

Tryptophan synthetase(trpB and A)

N-(5’-Phosphoribosyl)-Anthranilate isomerase Indole-3’-glycerol phosphate synthetase

Tryptophan synthetase


The The TrpTrp Operon: Operon:When Tryptophan Is PresentWhen Tryptophan Is Present

STOPRight therePolymerase

Trp

Trp

Repressor

Repressor

Repressor Promo. trpD trpBLead.Operator trpAtrpCtrpEAten.RNAPol.

FoiledAgain!

Repressor mRNA



The The TrpTrp Operon: Operon:When Tryptophan Is AbsentWhen Tryptophan Is Absent

Repressor

Repressor Promo. trpD trpBLead.Operator trpAtrpCtrpEAten.

Repressor mRNA


RNAPol.

RNAPol.

Repressor needs hislittle buddy tryptophan if

I’m to be stoppedI need

tryptophan


AttenuationAttenuationThe trp operon is controlled both by a repressor and

attenuationAttenuation is a mechanism that works only because

of the way transcription and translation are coupled in prokaryotes

Therefore, to understand attenuation, it is first necessary to understand transcription and translation in prokaryotes


3’

5’

5’

3’

Transcription And Translation Transcription And Translation In ProkaryotesIn Prokaryotes

Ribosome

Ribosome5’

mRNA

RNAPol.


Met-Lys-Ala-Ile-Phe-Val-AAGUUCACGUAAAAAGGGUAUCGACA-AUG-AAA-GCA-AUU-UUC-GUA-

Leu-Lys-Gly-Trp-Trp-Arg-Thr-Ser-STOPCUG-AAA-GGU-UGG-UGG-CGC-ACU-UCC-UGA-AACGGGCAGUGUAUU

CACCAUGCGUAAAGCAAUCAGAUACCCAGCCCGCCUAAUGAGCGGGCUUUU

Met-Gln-Thr-Gln-Lys-ProUUUU-GAACAAAAUUAGAGAAUAACA-AUG-CAA-ACA-CAA-AAA-CCG trpE . . .Terminator

The The TrpTrp Leader and Leader and AttenuatorAttenuator

4

1 2

3


The mRNA Sequence Can The mRNA Sequence Can Fold In Two WaysFold In Two Ways

4

1 23

Terminatorhairpin

4

1 2

3


3’

5’

5’

3’

The Attenuator The Attenuator When Starved For TryptophanWhen Starved For Tryptophan

41

23

RNAPol.

Ribosome

Leader peptide

Ribosome stalls over sequence 1 and sequence 2 binds to sequence 3 preventing formation of the 3, 4 hairpin

Help,I need

Tryptophan


3’

5’

5’

3’

The Attenuator The Attenuator When Tryptophan Is PresentWhen Tryptophan Is Present

4

1 2

3

RNAPol.

Ribosome

Leader peptide is released

Ribosome passes over sequence 1 and onto sequence 2 allowing sequence 3 to form the 3, 4 hairpin


3’

5’

5’

3’


RNAPol.

Ribosome

The 3, 4 hairpin destabilizes the elongation complex

4

1 2

3


3’

5’

5’

3’


Ribosome

RNAPol.

RNA polymerase falls off ending transcription

4

1 2

3


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©2000 Timothy G. Standish Ecclesiastes 3:1, 17 1To every thing there is a season, and a time to...

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