Controlling Controlling Gene ExpressionGene Expression

Timothy 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 genes are called constitutive genes and are expressed by all cells.

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

For example, beta cells in the pancreas make the protein insulin by expressing the insulin gene. If neurons expressed insulin, problems would result.

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 Lactose is a sugar that E. coli is unlikely to encounter, 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 The four genes are:

– LacZ - -galactosidase - An enzyme that hydrolizes the bond between galactose and glucose

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

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

– 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: Promoter

– These are specific DNA sequences to which RNA Polymerase binds so that transcription can occur

– The lac operon promoter also has a binding site for another protein called CAP

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 Promoter LacY LacALacZOperatorCAPBinding

Repressor

Repressor mRNA

Hey man, I’m constitutive

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 Promoter LacY LacALacZOperatorCAPBinding

Repressor

Repressor mRNA

Hey man, I’m constitutive

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

Repressor Promoter LacY LacALacZOperatorCAPBinding

CAPcAMP

CAPcAMP

CAPcAMP

Bind to mePolymerase

RNAPol.

Repressor

Repressor mRNA

Hey man, I’m constitutive

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 Enzymes encoded by genes in the trp operon are all

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

Antrhanilate

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

Hey man, I’m constitutive

AttenuationAttenuation

The trp operon is controlled both by a repressor and attenuation

Attenuation 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

Help,I need

Tryptophan

3’

5’

5’

3’

The Attenuator The Attenuator When Tryptophan Is PresentWhen Tryptophan Is Present

41

23

RNAPol.

Ribosome

RNAPol.

Control Of Expression In Control Of Expression In EukaryotesEukaryotes

Some of the general methods used to control expression in prokaryotes are used in eukaryotes, but nothing resembling operons is known

Eukaryotic genes are controlled individually and each gene has specific control sequences preceding the transcription start site

In addition to controlling transcription, there are additional ways in which expression can be controlled in eukaryotes

Eukaryotes Have Large Eukaryotes Have Large Complex GenomesComplex Genomes

The human genome is about 3 x 109 base pairs or ≈ 1 m of DNA

Because humans are diploid, each nucleus contains 6 3 x 109 base pairs or ≈ 2 m of DNA

That is a lot to pack into a little nucleus!

Eukaryotic DNA Must be Eukaryotic DNA Must be PackagedPackaged

Eukaryotic DNA exhibits many levels of packaging

The fundamental unit is the nucleosome, DNA wound around histone proteins

Nucleosomes arrange themselves together to form higher and higher levels of packaging.

Highly Packaged DNA Cannot Highly Packaged DNA Cannot be Expressedbe Expressed

The most highly packaged form of DNA is “heterochromatin”

Heterochromatin cannot be transcribed, therefore expression of genes is prevented

Chromosome puffs on some insect chomosomes illustrate where active gene expression is going on

Only a Subset of Genes is Only a Subset of Genes is Expressed at any Given TimeExpressed at any Given Time

It takes lots of energy to express genes Thus it would be wasteful to express all genes all the time By differential expression of genes, cells can respond to

changes in the environment Differential expression allows cells to specialize in

multicelled organisms. Differential expression also allows organisms to develop

over time.

DNA

Cytoplasm

Nucleus

G AAAAAA

Export

Degradation etc.G AAAAAA

Control of Gene ExpressionControl of Gene Expression

G AAAAAA

RNAProcessing

mRNA

RNA

Transcription

Nuclear pores

Ribosom

e

Translation

Packaging

Modification

Transportation

Degradation

Logical Expression Control PointsLogical Expression Control Points DNA packaging Transcription RNA processing mRNA export mRNA masking/unmasking and/or

modification mRNA degradation Translation Protein modification Protein transport Protein degradation

Increasing costIncreasing cost

The logical place to control

expression is before the

gene is transcribed

The logical place to control

expression is before the

gene is transcribed

A “Simple” Eukaryotic GeneA “Simple” Eukaryotic Gene

Terminator Sequence

Promoter/Control Region

Transcription Start Site

3’5’

RNA Transcript

Introns

Exon 2 Exon 3Int. 2Exon 1 Int. 1

3’ Untranslated Region5’ Untranslated Region

Exons

5’DNA

3’

EnhancersEnhancers

Enhancer Transcribed Region

3’5’ TF TFTF

3’5’ TF TFTF

5’RNA

RNAPol.

RNAPol.

Many bases

Promoter

Eukaryotic mRNAEukaryotic mRNA

Protein Coding Region

3’ Untranslated Region5’ Untranslated Region

Exon 2 Exon 3Exon 1 AAAAAG 3’5’

3’ Poly A Tail5’ Cap

RNA processing achieves three things: Removal of introns Addition of a 5’ cap Addition of a 3’ tail

This signals the mRNA is ready to move out of the nucleus and may control its lifespan in the cytoplasm