Gene Regulation

How is it possible that each kind of cell looks and functions

differently from one another if all cells in your body contain the

same exact copy of DNA ?

Regulation of Gene Expression

Several Levels in Which to Control Gene Expression

1. Pre-transcriptional level2. Transcriptional level3. Post-transcriptional level4. Translational level5. Post-translational level

Signal

NUCLEUSChromatin

Chromatin modification:DNA unpacking involvinghistone acetylation and

DNA demethylationDNA

Gene

RNA Exon

Gene availablefor transcription

Transcription

Primary transcript

IntronRNA processing

TailmRNA in nucleus

Transport to cytoplasm

Cap

CYTOPLASM

Regulation in the Nucleus

CYTOPLASM

mRNA in cytoplasm

TranslationDegradationof mRNA

Polypeptide

Protein processing, suchas cleavage and

chemical modification

Active protein

Transport to cellulardestination

Degradationof protein

Cellular function(such as enzymaticactivity, structural support)

Regulation in the Cytoplas

m

Signal

NUCLEUSChromatin

Chromatin modification:DNA unpacking involvinghistone acetylation and

DNA demethylationDNA

Gene

RNA Exon

Gene availablefor transcription

Transcription

Primary transcript

IntronRNA processing

TailmRNA in nucleus

Transport to cytoplasm

CYTOPLASM

mRNA in cytoplasm

TranslationDegradationof mRNA

Polypeptide

Cap

Protein processing, suchas cleavage and

chemical modification

Active protein

Transport to cellulardestination

Degradationof protein

Cellular function(such as enzymaticactivity, structural support)

How does DNA packing in

eukaryotes help to regulate gene expression?

Pre-transcriptional Rebulation

Nucleosome

Unacetylated histones Acetylated histones

Histonetails

Unravelling of the Chromosome

Early embryo:

Two cellpopulationsin adult cat:

X chromosomes

Cell division andX chromosomeinactivation

Allele fororange fur

Allele forblack fur

Active X

Orange furBlack fur

InactiveXActive X

Tight packing of the

X chromosome in female mammals(X inactivation)

Demethylation of DNA

Transcriptional LevelA gene can be either turned “on” or “off”.

ON – the gene is expressed (i.e., the gene is transcribed and translated to form a protein)

 OFF – the gene is not expressed (i.e., no

transcription, no translation, no protein is formed)

 Each kind of cell turns on only specific genes

depending on its needs or function.

How does a cell turn on some genes and not

others?

By way of regulatory sites (operators) found “upstream” from a gene and regulatory proteins that bind to these sites.

A Typical Gene

Gene Regulation: An Example

The best understood gene regulation process is the control of expression of the lac operon.

The “lac operon” is a group of genes found in

E. coli (bacteria) that are expressed all at the same time under the control of the same promoter and the same regulatory site (operator).  

This operon contains three genes that code for proteins that allow bacteria to use lactose (a sugar) as a food source.

DNA

PromoterOperator

Regulatorygene

NoRNAmade

IacZlacI

mRNA RNApolymerase

3

5

ActiverepressorProtein

(a) Lactose absent, repressor active, operon off

IacZ IacY IacAIacI

DNA lac operon

Permease Transacetylase-Galactosidase

mRNA

Protein

RNA polymerase

mRNA 53

5

Inactiverepressor

Allolactose(inducer)

(b) Lactose present, repressor inactive, operon on

Repressor Protein

Promoter

Lactose

The lac genes are turned OFF by a

“repressor protein”

and

turned ON by the presence of lactose.

In general, E. coli uses glucose as a food source and the lac genes are not expressed. In this state a repressor protein binds to the “operator” upstream from the lac genes and inhibits the transcription of the these genes by preventing RNA polymerase to bind to the promoter.

However, when the bacterium comes in contact with lactose, some lactose molecules enter the cell and bind to the repressor protein which causes its release from the operator. This now allows RNA polymerase to bind to the promoter region and therefore allows for the genes to be expressed.

Lac vs. Trp Operon Regulation

(a) Tryptophan absent, repressor inactive, operon on

Polypeptide subunits that make upenzymes for tryptophan synthesis

Protein

Inactiverepressor

mRNA

5

3

E D C B A

Promoter

DNA

Regulatorygene

RNApolymerase

Promoter

trp operon

Genes of operon

OperatorStart codon Stop codon

mRNA 5

trpE trpD trpC trpB trpAtrpR

DNA

mRNA

Protein Activerepressor

No RNAmade

Tryptophan(corepressor)

(b) Tryptophan present, repressor active, operon off

Transcription Factors• In eukaryotic cells, the default state for most genes is

« off »

• For housekeeping genes the default state is « on »

• RNA polymerase needs « transcription factors » in order to attach itself to a promoter and therefore transcribe the DNA

• Transcription factors fall into two main categories; Activators and Repressors

• Activator proteins bind to sequences of DNA upstream or downstream from the promoter called enhancers

• Repressor proteins bind to sequences of DNA called silencers

DNAUpstream

Enhancer(distal control

elements)

Proximalcontrol

elementsTranscription

start site

Exon Intron Exon

Promoter

Intron Exon

Poly-A signalsequence

Transcriptiontermination

region

Down-stream

Transcription

Exon Intron IntronExon Exon

Poly-Asignal

Primary RNAtranscript(pre-mRNA)

5 Cleaved 3end ofprimarytranscript

Intron RNA

mRNA

RNA processing

Coding segment

3

5 5 3Cap UTRStart

codonStop

codon UTR Poly-Atail

G P P P AAAAAA

Activationdomain

DNA

DNA-bindingdomain

DNA

EnhancerDistal controlelement

Activators PromoterGene

TATA box

DNA

EnhancerDistal controlelement

Activators PromoterGene

TATA box

DNA-bendingprotein

Group of mediator proteins

General transcriptionfactors

DNA

EnhancerDistal controlelement

Activators PromoterGene

TATA box

DNA-bendingprotein

Group of mediator proteins

General transcriptionfactors

RNApolymerase II

RNApolymerase II

RNA synthesisTranscriptioninitiation complex

Differentiation

• Results from selective gene expression

• Example– Muscle cells – genes for creating actin and

myosin are turned on to create muscle fibres– Pancreatic cells – genes that encode glucagon

and insulin are turned on– Blood cells – genes encoding hemoglobin are

turned on

• Housekeeping genes – genes that are turned on in all cells that code for processes that all cells must undergo, like enzymes involved in glycolysis

Albumin gene

Crystallin gene

Promoter

Promoter

(b) LENS CELL NUCLEUS

Availableactivators

Albumin genenot expressed

Crystallin geneexpressed

Crystallin genenot expressed

Albumin geneexpressed

Availableactivators

(a) LIVER CELL NUCLEUS

Controlelements

Enhancer

Enhancer

Dedifferentiation in Plants

How do eukaryotic cells turn on or off the genes of related

functions located on different chromosomes?

• They all use the same group of transcription factors & enhancers & silencers

• This way the transcription factors will all bind at the same time and genes located in very different locations can be turned on or off simultaneously

PromoterReportergene

Enhancer with possiblecontrol elements

Relative level of reportermRNA (% of control)

1

0 50 150 200100

2 3

Regulation through RNA Splicing – Post-

transcriptional Regulation• mRNA cannot leave the nucleus

until splicing occurs since it has all of the splicing machinery attached to it and therefore cannot fit through the nuclear pores

• Alternative splicing leads to the creation of two or more proteins from just one gene

Other Regulators of Gene Expression

• Breakdown of mRNA• Minutes after transcription in prokaryotes

and hours to weeks in eukaryotes

• Initiation of Translation• Protein Activation

• Post-translational folding and cleavage

• Protein Breakdown• Damaged proteins break down to form new

ones• The breakdown of proteins allows the cell to

quickly adapt to the change environment

miRNAmiRNA-proteincomplex

Translation blockedmRNA degraded

The miRNA bindsto a target mRNA.

1

If bases are completely complementary, mRNA is degraded.If match is less than complete, translation is blocked.

2

Chromatin modification Transcription

RNA processing

Translation

mRNA degradation Protein processing and degradation

• Protein processing and degradation aresubject to regulation.

• Each mRNA has acharacteristic life span.

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

mRNA or

Primary RNAtranscript

• Alternative RNA splicing:

• The genes in a coordinately controlledgroup all share a combination of controlelements.

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

Bending ofthe DNAenablesactivators tocontact proteins atthe promoter, initiating transcription.

• Genes in highly compactedchromatin are generally nottranscribed.• Histone acetylation seemsto loosen chromatinstructure,enhancingtranscription.

• DNA methylation generallyreduces transcription.

Chromatin modification

Transcription

RNA processing

mRNAdegradation

Translation

Protein processingand degradation

Bozeman ScienceDevelopmental Biology &

Differential Gene Expression

Observing Differential Gene Expression

Using Quantitative PCR

Test tube containingreverse transcriptaseand mRNA

DNA in nucleus

mRNAs incytoplasm

1

Test tube containingreverse transcriptaseand mRNA

DNA in nucleus

mRNAs incytoplasm

Reverse transcriptasemakes the firstDNA strand.

Reversetranscriptase

mRNAPoly-A tail

DNAstrand

Primer

53

35

A A A A A AT

1

2

T T T T

Test tube containingreverse transcriptaseand mRNA

DNA in nucleus

mRNAs incytoplasm

Reverse transcriptasemakes the firstDNA strand.

Reversetranscriptase

mRNAPoly-A tail

DNAstrand

Primer

53

35

A A A A A A

1

2

mRNA is degraded.353

35

A A AT

A A A

T T T T T

T T T T

Test tube containingreverse transcriptaseand mRNA

DNA in nucleus

mRNAs incytoplasm

Reverse transcriptasemakes the firstDNA strand.

Reversetranscriptase

mRNAPoly-A tail

DNAstrand

Primer

53

35

A A A A A A

1

2

mRNA is degraded.353

35

A A A A A A

DNA polymerasesynthesizes thesecond strand.

DNApolymerase

53

35

4

T

T T T T T

T T T T

Test tube containingreverse transcriptaseand mRNA

DNA in nucleus

mRNAs incytoplasm

Reverse transcriptasemakes the firstDNA strand.

Reversetranscriptase

mRNAPoly-A tail

DNAstrand

Primer

53

35

A A A A A A

1

2

mRNA is degraded.353

35

A A A A A A

DNA polymerasesynthesizes thesecond strand.

DNApolymerase

53

35

4

53

35

cDNA

cDNA carries completecoding sequencewithout introns.

5

T

T T T T T

T T T T

mRNAs

cDNAs

Embryonic stages1

cDNA synthesis

PCR amplification

Gel electrophoresis

Results

Technique

1

2

3

Primers

-globingene

2 3 4 5 6

Observing Differential Gene Expression

Using DNA chips or Microarrays

Genes in redwells expressedin first tissue.

Genes in greenwells expressedin second tissue.

Genes in yellowwells expressedin both tissues.

Genes in blackwells notexpressed in either tissue.

DNA microarray

The Genetic Control of Embryonic Development

Gene Expression in Embryogenesis

• Embryogenesis results from cascades of gene expression that allow for cell-to-cell signaling that direct embryonic development

• Homeotic genes are master control genes that regulate batteries of other genes which will, in turn, determine the anatomy of parts of the body

• This genetic approach to the study of embryogenesis has revolutionized developmental biology

• Most of the what we know about embryogenesis comes from studies performed on fruit flies and a nematode called C. elegans

Regulation of Gene

Expression in

Drosophila

Signal Transduction Pathways & Gene

Expression• How do adjacent cells communicate?

• Through signal transduction pathways (« a series of molecular changes that conversts a signal on a target cell’s surface to a specific response inside the cell »)

• Signal proteins from one cell will cause gene expression in adjacent cells

• This type of communication is key to embryonic development and the coordination of cellular activities within a mature organism

Homeotic Genes are Ancient

• All homeotic genes contain a common sequence of 180 nucleotides called homeoboxes

• These homeoboxes code for a 60-amino acid polypeptide chain in different homeotic proteins and enable them to bind to DNA and either turn on or turn off genes involved in embryonic development

• These homeoboxes can be found in virtually every eukaryotic organism and have even been seen in some prokaryotic cells

• The presence of homeoboxes in such a variety of organisms supports the idea that all life came from the same ancestor

• The fact that these ancient genes are control genes underscores the importance of gene regulation in living organisms

Figure 16.15

Culturedstem cells

Embryonicstem cells

Liver cells Nerve cells Blood cells

Adultstem cells

Differentcultureconditions

Differenttypes ofdifferentiatedcells

Cells that can generateall embryonic cell types

Cells that generate a limitednumber of cell types

Figure 16.14

Stem cell

Stem cell Precursor cell

Fat cells

Celldivision

and

or orBone cells Whiteblood cells

Genetic Basis of Cancer

Cancer is the Result of Multiple Mutations

Cancers are Triggered by the Deregulation of Cell

Division• The deregulation of cell division in

cancerous cells is due to mutations in genes that control the cell cycle

• Our earliest understanding of cancer came from the discovery of a virus that causes cancer in chickens

• This virus carries an oncongene (potentially cancer-causing gene) that is a mutated version of normal gene found in chickens

Proto-oncogene

Translocationor transposition:gene moved tonew locus, undernew controls

Normal growth-stimulating proteinin excess

Newpromoter

Oncogene

Gene amplification:multiple copies ofthe gene

Proto-oncogene

Normal growth-stimulating proteinin excess

within a controlelement

Proto-oncogene

Point mutation:

withinthe gene

OncogeneOncogene

Normal growth-stimulating protein in excess

Hyperactive ordegradation-resistantprotein

within a controlelement

Proto-oncogene

Gene amplification:multiple copies ofthe gene

Proto-oncogene Proto-oncogene

Point mutation:

withinthe gene

Translocationor transposition:gene moved tonew locus, undernew controls

Normal growth-stimulating proteinin excess

Newpromoter

OncogeneOncogeneOncogene

Normal growth-stimulating proteinin excess

Normal growth-stimulating protein in excess

Hyperactive ordegradation-resistantprotein

Growth factor

G protein

Receptor

Proteinkinases

NUCLEUSTranscriptionfactor (activator)

Protein thatstimulatesthe cell cycle

NUCLEUSTranscriptionfactor (activator)

Overexpressionof protein

MUTATION

Ras protein active withor without growth factor.

GTP

Ras

GTP

Ras

1

2

3

4

5 6

Tumor-Suppressor Genes

Protein kinases

NUCLEUS

DNA damagein genome

Defectiveor missingtranscriptionfactor

MUTATIONUVlight

1

2

3

Inhibitoryproteinabsent

DNA damagein genome

UVlight

Active formof p53

Protein thatinhibits thecell cycle

Colon

Loss of tumor-suppressor geneAPC (or other)

Loss oftumor-suppressor gene p53

Activation ofras oncogene

Colon wallNormal colonepithelial cells

Small benigngrowth(polyp)

Loss of tumor-suppressor gene DCC

Malignanttumor(carcinoma)

Larger benigngrowth(adenoma)

Additionalmutations

1 2

3

4

5

Loss of tumor-suppressor geneAPC (or other)

Loss oftumor-suppressor gene p53

Activation ofras oncogene

Colon wall

Normal colonepithelial cells

Small benigngrowth (polyp)

Loss of tumor-suppressor gene DCC

Malignant tumor(carcinoma)

Larger benigngrowth (adenoma)

Additionalmutations

1

2

3

4

5

Animal Cloning

Nuclear Transplantation

• First performed in the 50’s with frogs

• Replacement of the nucleus of an egg cell or zygote cell with the nucleus of an adult somatic cell

• This process can be used for two main purposes:

1. Reproductive Cloning2. Therapeutic Cloning with the production of

ES cells (embryonic stem cells)

Grown in culture

Results

Cell cyclearrested,causing cells todedifferentiate

Implanted in uterusof a third sheep

Culturedmammarycells

Embryonicdevelopment

TechniqueMammarycell donor

Egg cell donor

Egg cell from ovary

Nucleusremoved

Nucleus from mammary cell

Surrogatemother

Cells fused

Early embryo

Lamb (“Dolly”)genetically identical tomammary cell donor

1 2

3

4

5

6

Reproductive Cloning• Has been done on frogs, sheep, mice, cats, cows,

pigs, mules and monkeys!

Benefits: 1. Farmers create a identical herds with desired traits2. Pharmaceutical companies can use certain cloned

mammals to produce pharmaceutical drugs3. Cloned pigs missing one of the proteins that cause

immunal rejection in human transplant patients, may one day serve as transplant donors

4. Help repopulating endangered species? 5. Genetic research?

Disadvantages:1. Ethics2. Goes against evolution (decreases variation)

Therapeutic Cloning

• Insert a patient’s nucleus into an enucleated ES cell so that the patient won’t reject the new cells because they will be genetically identical

• Problem: ES cells come from destroying human embryos

• Alternative: Use bone marrow cells, but they are not totipotent