Lecture 26

Regulation of gene expression

Signal

NUCLEUSChromatin

Chromatin modification:DNA unpacking involvinghistone acetylation and

DNA demethylationDNA

Gene

Gene availablefor transcription

RNA ExonPrimary transcript

Transcription

Intron

Cap

Tail

mRNA in nucleus

Transport to cytoplasm

CYTOPLASM

mRNA in cytoplasm

TranslationDegradationof mRNA

Polypeptide

Protein processing, suchas cleavage and

chemical modification

Active proteinDegradation

of proteinTransport to cellular

destination

Cellular function (suchas enzymatic activity,structural support)

Overview• Prokaryotes and eukaryotes alter gene expression in

response to their changing environment• In multicellular eukaryotes, gene expression regulates

development and is responsible for differences in cell types

• RNA molecules play many roles in regulating gene expression in eukaryotes

Regulation of gene expression

in prokaryotic cell - Operon units, system of

negative feedback

in eukaryotic cell come at any stage of gene

expression and proteosynthesis. Important are

noncoding RNAs.

Bacteria often respond to environmental change by regulating

transcription• Natural selection has favored bacteria that produce only

the products needed by that cell• A cell can regulate the production of enzymes by

feedback inhibition or by gene regulation• Gene expression in bacteria is controlled by the operon

model

Precursor

Feedbackinhibition

Enzyme 1

Enzyme 2

Enzyme 3

Tryptophan

Regulation of enzymeactivity

Regulation of enzymeproduction

Regulationof geneexpression

trpE gene

trpD gene

trpC gene

trpB gene

trpA gene

Operons: The Basic Concept• A cluster of functionally related genes can be under

coordinated control by a single “on-off switch”• The regulatory “switch” is a segment of DNA called an

operator usually positioned within the promoter• An operon is the entire stretch of DNA that includes

the operator, the promoter, and the genes that they control

Operon is a functional unit common in bacteria and phages. Activation

and inhibition of transcription are regulated in response of conditions

in environment.

Prokaryotic genetic information is not divided into introns and exons.

Operon

• is coordinately regulated clusters of genes,

which are transcribed into one mRNA

(polygenic mRNA)

• are genes for particular metabolic pathway

and are regulated by common promoter and

are ordered on DNA following each other

Escherichia coli

Lac operon, Trp operon – model systems =

metabolic pathways of

• utilization of lactose gen lacZ, lacY, lacA, catabolic

pathway with negative and positive regulation

• enzymes for TRP synthesis, anabolic pathway

with negative regulation

• The operon can be switched off by a protein repressor• The repressor prevents gene transcription by binding to

the operator and blocking RNA polymerase• The repressor can be in an active or inactive form,

depending on the presence of other molecules• A corepressor is a molecule that cooperates with a

repressor protein to switch an operon off• For example, E. coli can synthesize the amino acid

tryptophan

• By default the trp operon is on and the genes for tryptophan synthesis are transcribed

• When tryptophan is present, it binds to the trp repressor protein, which turns the operon off

• The repressor is active only in the presence of its corepressor--tryptophan; thus the trp operon is turned off (repressed) if tryptophan levels are high

Promoter

DNA

Regulatory gene

mRNA

trpR

5

3

Protein Inactive repressor

RNApolymerase

Promoter

trp operon

Genes of operon

Operator

mRNA 5

Start codon Stop codon

trpE trpD trpC trpB trpA

E D C B A

Polypeptide subunits that make upenzymes for tryptophan synthesis

(a) Tryptophan absent, repressor inactive, operon on

(b) Tryptophan present, repressor active, operon off

DNA

mRNA

Protein

Tryptophan (corepressor)

Activerepressor

No RNAmade

Repressible and Inducible Operons: Two Types of Negative Gene Regulation

• A repressible operon is one that is usually on; binding of a repressor to the operator shuts off transcription

• The trp operon is a repressible operon• An inducible operon is one that is usually off; a

molecule called an inducer inactivates the repressor and turns on transcription

• The lac operon is an inducible operon and contains genes that code for enzymes used in the hydrolysis and metabolism of lactose

• By itself, the lac repressor is active and switches the lac operon off

• A molecule called an inducer inactivates the repressor to turn the lac operon on

(a) Lactose absent, repressor active, operon off

(b) Lactose present, repressor inactive, operon on

Regulatorygene

Promoter

Operator

DNA lacZlacI

lacI

DNA

mRNA5

3

NoRNAmade

RNApolymerase

ActiverepressorProtein

lac operon

lacZ lacY lacADNA

mRNA

5

3

Protein

mRNA 5

Inactiverepressor

RNA polymerase

Allolactose(inducer)

-Galactosidase Permease Transacetylase

Lac operon - negative regulation

• regulatory gene produces repressor, which

binds operator and causes that RNAP is not

able to initialize transcription

• in the presence of lactose repressor is released

from operator. The repressor is changed by

inducer / lactoseRNA polymerase starts the transcription. In 2-3 minutes the amount of

enzymes is increased 1000x

• Inducible enzymes usually function in catabolic pathways; their synthesis is induced by a chemical signal

• Repressible enzymes usually function in anabolic pathways; their synthesis is repressed by high levels of the end product

• Regulation of the trp and lac operons involves negative control of genes because operons are switched off by the active form of the repressor

Positive Gene Regulation

• Some operons are also subject to positive control through a stimulatory protein, such as catabolite activator protein (CAP), an activator of transcription

• When glucose (a preferred food source of E. coli) is scarce, CAP is activated by binding with cyclic AMP (cAMP)

• Activated CAP attaches to the promoter of the lac operon and increases the affinity of RNA polymerase, thus accelerating transcription

• In the presence of glucose, E. coli preferentially uses

glucose for decomposing.

• If is low level of glucosis, the cAMP is increased.

• CAP (Catabolite activator protein) in the presence

of cAMP attaches promoter and activates the

transcription.

• CAP is allosteric regulatory protein• When glucose levels increase, CAP detaches from the

lac operon, and transcription returns to a normal rate

Lac operon - positive regulation

Promoter

DNA

CAP-binding site

lacZlacI

RNApolymerasebinds andtranscribes

Operator

cAMPActiveCAP

InactiveCAP

Allolactose

Inactive lacrepressor

(a) Lactose present, glucose scarce (cAMP level high):abundant lac mRNA synthesized

Promoter

DNA

CAP-binding site

lacZlacI

OperatorRNApolymerase lesslikely to bind

Inactive lacrepressor

InactiveCAP

(b) Lactose present, glucose present (cAMP level low):little lac mRNA synthesized

Summary

each operon consists of • promoter (for RNA polymerase)• operator (for repressor)• several structural genes• terminator

repressor = allosteric protein encoded by regulatory gene

co-repressor = product moleculeinducer = substrate molecule

Eukaryotic gene expression is regulated at many stages

• All organisms must regulate which genes are expressed at any given time

• In multicellular organisms regulation of gene expression is essential for cell specialization

Gene expression of eukaryotic cells

• each cell maintains specific program /

differential gene expression

• one mRNA carries information for one gene

(monogennic mRNA)

• posttranscription modifications of RNA

RNA processing and splicing

• regulation system is performed at the several

levels = transcription, translation, protein

activation + secretion

Differential Gene Expression

• Almost all the cells in an organism are genetically identical

• Differences between cell types result from differential gene expression, the expression of different genes by cells with the same genome

• Abnormalities in gene expression can lead to diseases including cancer

• Gene expression is regulated at many stages

Signal

NUCLEUSChromatin

Chromatin modification:DNA unpacking involvinghistone acetylation and

DNA demethylationDNA

Gene

Gene availablefor transcription

RNA ExonPrimary transcript

Transcription

Intron

Cap

Tail

mRNA in nucleus

Transport to cytoplasm

CYTOPLASM

mRNA in cytoplasm

TranslationDegradationof mRNA

Polypeptide

Protein processing, suchas cleavage and

chemical modification

Active proteinDegradation

of proteinTransport to cellular

destination

Cellular function (suchas enzymatic activity,structural support)

Many steps at which eucaryotic gene expression can be controlled

• chromatin changes

• transcription

• processing RNA

• transport to cytoplasm

• degradation of mRNA

• translation

• cleavage, chemical modification

• protein degradation

more complicated regulating system

• Heterochromatin is highly condensed that is why

transcriptional enzymes can not reach the DNA

• Acetylation / deacetylation of histons

• Methylation [cytosin] - inactive DNA is highly

methylated

DNA methylation and histone de-acetylation repress

the transcription.

1. Chromatin changes

• DNA methylation

is esential for long-term inactivation of genes during

cell differentiation

Gene imprinting in mamals

• methylation constantly turns off the maternal or the

paternal allele of a gene in early development

• certain genes are expressed in a parent-of-origin-

specific manner

Epigenetic inheritance

Histone Modifications• In histone acetylation, acetyl groups are attached to

positively charged lysines in histone tails• This loosens chromatin structure, thereby promoting the

initiation of transcription• The addition of methyl groups (methylation) can

condense chromatin; the addition of phosphate groups (phosphorylation) next to a methylated amino acid can loosen chromatin

Amino acidsavailablefor chemicalmodification

Histone tails

DNA double helix

Nucleosome(end view)

(a) Histone tails protrude outward from a nucleosome

Unacetylated histones Acetylated histones

(b) Acetylation of histone tails promotes loose chromatinstructure that permits transcription

DNA Methylation• DNA methylation, the addition of methyl groups to

certain bases in DNA, is associated with reduced transcription in some species

• DNA methylation can cause long-term inactivation of genes in cellular differentiation

• In genomic imprinting, methylation regulates expression of either the maternal or paternal alleles of certain genes at the start of development

Epigenetic Inheritance• Although the chromatin modifications just discussed do

not alter DNA sequence, they may be passed to future generations of cells

• The inheritance of traits transmitted by mechanisms not directly involving the nucleotide sequence is called epigenetic inheritance

Regulation of Transcription Initiation

• Chromatin-modifying enzymes provide initial control of gene expression by making a region of DNA either more or less able to bind the transcription machinery

2. Transcription

Transcription factors:

proteins that bind DNA and facilitate or inhibit RNA

polymerase to bind. They are a part of transcription initiation

complex.

general transcription factors for all protein-coding genes

specific transcription factors – transcription of particular

genes at appropriate time and place

- enhancers, activators, inhibitors, repressors

Organization of a Typical Eukaryotic Gene

• Associated with most eukaryotic genes are multiple control elements, segments of noncoding DNA that serve as binding sites for transcription factors that help regulate transcription

• Control elements and the transcription factors they bind are critical to the precise regulation of gene expression in different cell types

Enhancer(distal control

elements)

DNA

UpstreamPromoter

Proximalcontrol

elementsTranscription

start site

Exon Intron Exon ExonIntron

Poly-Asignal

sequenceTranscriptiontermination

region

DownstreamPoly-Asignal

Exon Intron Exon ExonIntron

Transcription

Cleaved3 end ofprimarytranscript

5Primary RNAtranscript(pre-mRNA)

Intron RNA

RNA processing

mRNA

Coding segment

5 Cap 5 UTRStart

codonStop

codon 3 UTR

3

Poly-Atail

PPPG AAA AAA

The Roles of Transcription Factors• To initiate transcription, eukaryotic RNA polymerase

requires the assistance of proteins called transcription factors

• General transcription factors are essential for the transcription of all protein-coding genes

• In eukaryotes, high levels of transcription of particular genes depend on control elements interacting with specific transcription factors

• Proximal control elements are located close to the promoter

• Distal control elements, groupings of which are called enhancers, may be far away from a gene or even located in an intron

Enhancers and Specific Transcription Factors

• An activator is a protein that binds to an enhancer and stimulates transcription of a gene

• Activators have two domains, one that binds DNA and a second that activates transcription

• Bound activators facilitate a sequence of protein-protein interactions that result in transcription of a given gene

• Some transcription factors function as repressors, inhibiting expression of a particular gene by a variety of methods

• Some activators and repressors act indirectly by influencing chromatin structure to promote or silence transcription

ActivatorsDNA

EnhancerDistal controlelement

PromoterGene

TATA box

Generaltranscriptionfactors

DNA-bendingprotein

Group of mediator proteins

RNApolymerase II

RNApolymerase II

RNA synthesisTranscriptioninitiation complex

Cell-type specific transcription:

Genes encoding the enzymes of one metabolic

pathway are scattered over the different

chromosomes - coordinated control in

response of chemical signals from outside

the cell. The cell accept signals by receptors.

Signal transduction pathways activate

transcription activators or repressors.

Signal transduction pathways

Controlelements

Enhancer Promoter

Albumin gene

Crystallingene

LIVER CELLNUCLEUS

Availableactivators

Albumin geneexpressed

Crystallin genenot expressed

(a) Liver cell

LENS CELLNUCLEUS

Availableactivators

Albumin genenot expressed

Crystallin geneexpressed

(b) Lens cell

Coordinately Controlled Genes in Eukaryotes

• Unlike the genes of a prokaryotic operon, each of the co-expressed eukaryotic genes has a promoter and control elements

• These genes can be scattered over different chromosomes, but each has the same combination of control elements

• Copies of the activators recognize specific control elements and promote simultaneous transcription of the genes

Nuclear Architecture and Gene Expression

• Loops of chromatin extend from individual chromosomes into specific sites in the nucleus

• Loops from different chromosomes may congregate at particular sites, some of which are rich in transcription factors and RNA polymerases

• These may be areas specialized for a common function

Chromosometerritory

Chromosomes in theinterphase nucleus

Chromatinloop

Transcriptionfactory

10 m

Mechanisms of Post-Transcriptional Regulation

• Transcription alone does not account for gene expression

• Regulatory mechanisms can operate at various stages after transcription

• Such mechanisms allow a cell to fine-tune gene expression rapidly in response to environmental changes

Exons

DNA

Troponin T gene

PrimaryRNAtranscript

RNA splicing

ormRNA

1

1

1 1

2

2

2 2

3

3

3

4

4

4

5

5

5 5

• In alternative RNA splicing, different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns

3. Processing RNA

• The life span of mRNA molecules in the cytoplasm is a key to determining protein synthesis

• Eukaryotic mRNA is more long lived than prokaryotic mRNA

• Nucleotide sequences that influence the lifespan of mRNA in eukaryotes reside in the untranslated region (UTR) at the 3 end of the molecule

4, 5. transport of mRNA / degradation

6. Translation

At the initiation stage – regulatory proteins bind the

5’ end of the mRNA with the cap.

Activation or inactivation of protein factors to initiate

translation

7. Cleavage, chemical modifications

Cleavage

Post-translational modifications

Regulatory proteins [products] are activated

or inactivated by the reversible addition of

phosphate groups / phosphorylation

Sugars on surface of the cell / Glycosylation

• Polypeptide chain may

be cleaved into two or

three pieces

• Preproinsulin

• Proinsulin - disulfide

bridges

• Insulin

• Secretory protein

Acid/base - act/inact

Hydrolysis – localization, act/inact

Acetylation - act/inact

Phosphorylation - act/inact

Prenylation - localization

Glycosylation - targeting

Post-translational modifications

• Lifespan of protein is strictly regulated• Proteins are produced and degraded continually in

the cell.• Proteins to be degraded are tagged with ubiquitin.• Degradation of proteins marked with ubiquitin

occurs at the proteasome.

8. protein degradation

Chromatin modification

• Genes in highly compactedchromatin are generally nottranscribed.• Histone acetylation seemsto loosen chromatin structure,enhancing transcription.

• DNA methylation generallyreduces transcription.

mRNA degradation

• Each mRNA has a characteristic life span,determined in part bysequences in the 5 and3 UTRs.

• Regulation of transcription initiation:DNA control elements in enhancers bindspecific transcription factors.

Bending of the DNA enables activators tocontact proteins at the promoter, initiatingtranscription.

• Coordinate regulation:Enhancer for

liver-specific genesEnhancer for

lens-specific genes

Transcription

RNA processing

• Alternative RNA splicing:

Primary RNAtranscript

mRNA or

• Initiation of translation can be controlledvia regulation of initiation factors.

• Protein processing anddegradation by proteasomesare subject to regulation.

Translation

Protein processing and degradation

Chromatin modification

Transcription

RNA processing

mRNAdegradation

Translation

Protein processingand degradation

Example of gene regulation

Regulation & DevelopmentRegulation & Development

• hox Geneshox Genes– Control Organ & Tissue Development Control Organ & Tissue Development

In The EmbryoIn The Embryo

– Mutations Lead To Major ChangesMutations Lead To Major Changes• Drosophila With Legs In Place of Drosophila With Legs In Place of

AntennaeAntennae

Regulation & DevelopmentRegulation & Development

Regulation & DevelopmentRegulation & Development

hox Geneshox Genes Present In All EukaryotesPresent In All Eukaryotes– Shows Common AncestryShows Common Ancestry

– Pax 6 hox genePax 6 hox gene• Controls eye growth in Drosophila, Mice & Controls eye growth in Drosophila, Mice &

ManMan

• Pax 6Pax 6 from Mouse Placed In Knee from Mouse Placed In Knee Development Sequence Of Drosophila Development Sequence Of Drosophila Developed Into Eye Tissue.Developed Into Eye Tissue.

Common Ancestor >600M Years AgoCommon Ancestor >600M Years Ago

Homeotic mutations transform one body part into another.

Wild type Mutant

Eye

AntennaLeg

The Regulation of Eukaryotic Gene Expression

..using the example of PEPCK

PEPCK

• This is an acronym for an enzyme• PhosphoEnol Pyruvate CarboxyKinase• This enzyme is ONLY regulated by gene

expression!• No allosteric activators, covalent

modification etc• No activation by cAMP, inhibition by insulin

etc

PEPCK

• The enzyme is expressed in liver, kidney, adipose tissue and to a lesser extent in muscle

• It is a key enzyme in gluconeogenesis (the synthesis of new glucose, usually from lactate, pyruvate or alanine) and glyceroneogenesis (the synthesis of glycerol, usually from lactate, pyruvate or alanine)

PEPCK overexpression in muscle

• a mouse with PEPCK overexpressed in muscle only.

• This mouse was leaner than wild type mice, ran for longer and lived longer!

• They were also more aggressive.

• The overexpression had switched the muscle fuel usage to fatty acids with little lactate production.

The Supermouse….

• Eats 60% more food than wild type mice

• Weighs 40% less than wild type mice

• Can run for >4 h until exhaustion whereas the control littermates stop after only 10 min

• Has 2 – 3 fold less adipose tissue

PEPCK overexpression in adipose tissue

• A mouse has the PEPCK enzyme overexpressed in adipose tissue.

• The results couldn’t be further from supermouse!

PEPCK overexpression in fat cells

PEPCK overexpression in adipose tissue

• These mice are obese although metabolically healthy (as measured by glucose

tolerance and insulin sensitivity) until you put them on a high fat diet.

• Then you see insulin resistance and diabetes emerging.

PEPCK overexpression in liver

• Leads to altered glucose tolerance

• Insulin resistance

• Increased gluconeogenesis causes increased hepatic glucose production which is released into the blood stream

• This caused increased insulin secretion but ultimately insulin resistance.

PEPCK Knock out in liver

• Surprisingly these mice can maintain blood glucose under starvation conditions

• They develop liver steatosis (fatty livers) probably because of impaired oxidation of fatty acids

• A total PEPCK knock out in all tissues is lethal…mice die within days of birth.

Why the dramatically different outcome for the mouse when PEPCK is overexpressed in different tissues?It is after all the same enzyme catalysing

the same reaction.

Glyceroneogenesis

PEPcarboxykinase

COOH

C

CH2

O

CO

H2C COOH

COOH

oxaloacetateOAA

PO3

Phosphoenol pyruvatePEP

COOH

C O

CH3

COOH

HC OH

CH3

NADH NAD+

Pyruvate Lactate

CO2

Pyruvate Carboxylase LDH

CO2

GTP

GDPAlanine

Glyceroneogenesis

CH2OPO3

C O

CH2OH

CH2OPO3

HC OH

C

O

Glyceraldehyde 3-P

Dihydroxyacetone phosphate (DHAP)

H

CH2OPO3

HC OH

CH2OH

Glycerol 3-P

Triglycerides

Fatty acids H3C CO

S-CoA

PEP