Chapter 15: Regulation of Gene Expression - PowerPoint Presentation

Slide1

Chapter 15:

Regulation of Gene Expression

Anablep

anablep

with its “4 eyes”. Upper half of lids look aerially while the lower half looks into the water.

Cells of the two parts of the eye exhibit differential gene expressionSlide2

Individual bacteria respond to environmental change by regulating their gene expression

Express only genes whose products may be needed presently by the cell. (Don’t want to waste energy)

E. coli

lives in the human colon & can fine-tune its metabolism to the changing environment and food sourcesIf tryptohan is lacking, the cell responds by activating a gene that starts a metabolic pathway to produce tryptophan from another food source that is present.If tryptophan is plentiful, the bacteria stop making tryptophan to conserve energy.Slide3

Figure 18.20a, b

(a) Regulation of enzyme

activity

Enzyme 1

Enzyme 2

Enzyme 3

Enzyme 4

Enzyme 5

Regulation

of gene

expression

Feedback

inhibition

Tryptophan

Precursor

(b) Regulation of enzyme

production

Gene 2

Gene 1

Gene 3

Gene 4

Gene 5

–

–

This metabolic control occurs on two levels

Adjusting the activity of enzymes

already present. Sensitivity to chemical clues

When there is a build up of tryptophan, it shuts off the production of Enzyme 1

Short term response

Regulating the genes

encoding the metabolic enzymes

Tryptophan can shut down the transcription of a gene is a longer term responseSlide4

Operons

: The Basic Concept

In bacteria

, genes are often

clustered into groups of genes (such as the 5 for tryptophan) called operons, composed of:An operator, an “on-off” switch

which is a segment of DNA that

lies between

promoter

and enzyme-coding genesA promoter – 1 for the entire operonGenes for metabolic enzymes AnimationSlide5
Slide6

Animation

The Trp operon

Is usually turned “O

n

” (with no repressor protein stopping transcription)Can be switched off by a protein called a repressor proteintrpR is a regulatory gene which is continually expressed in small amounts. It produces the repressor protein that binds to Operator to prevent transcription

Figure 18.21a

Tryptophan absent,

repressor

inactive

,

operon

on. RNA polymerase attaches to the DNA at the promoter and transcribes the operon’s genes.

Genes of operon

Inactiverepressor

ProteinOperator

Polypeptides that make upenzymes for tryptophan synthesis

Promoter

Regulatory

gene

RNA

polymerase

Start codon Stop codon

Promoter

trp

operon

5

3

mRNA 5

trpD

trpE

trpC

trpB

trpA

trpR

DNA

mRNA

E

D

C

B

ASlide7

DNA

mRNA

Protein

Tryptophan

(

corepressor

)

Active

repressor

No RNA made

Tryptophan present

,

repressor

active

,

operon off

. As tryptophan accumulates, it inhibits its own production by activating the repressor protein. Tryptophan as a a corepressor

(b)RNA polymerase

trpR

Regulatorygene

Inactiverepressor

OperatorSlide8

Repressible and Inducible Operons: Two Types of Negative Gene Regulation

In a repressible operon

Transcription is usually on

but can be repressed when small amount of

Tryptophan binds to allosterically to regulatory proteinBinding of a specific repressor protein to the operator shuts off transcriptionTrp operon

In an

inducible

operon

Induced by a chemical signal

(

Allolactose in lac operon)Binding of an inducer to an innately inactive repressor inactivates the repressor and turns on transcriptionSlide9

Figure 18.22a

DNA

mRNA

Protein

Active

repressor

RNA

polymerase

No

RNA

made

lacZ

lacl

Regulatory

gene

Operator

Promoter

Lactose absent,

repressor active

,

operon

off

.

The lac repressor is innately active, and in the absence of lactose it switches off the operon by binding to the operator.

(a)

5

3

The

lac operon: regulated synthesis of Inducible enzymes. Regulates the catabolism of lactose to galactose and glucoseNeeds an inducer to remove repressor and start transcriptionanimationSlide10

mRNA 5'

DNA

mRNA

Protein

Allolactose

(inducer)

Inactive

repressor

lacl

lacz

lacY

lacA

RNA

polymerase

Permease

Transacetylase

-Galactosidase

5

3

(b)

When Lactose is present

,

the

repressor is

inactive

,

operon

is on &

β –galactosidase is produced. Allolactose, an isomer of lactose, acts as the inducer by derepresses the operon by inactivating the repressor

.

In this way, the enzymes for lactose utilization are induced.

mRNA 5

lac

operonSlide11

No lactose present, no need to produce enzymes to hydrolyze lactose to glucose &

galactose

If Lactose is present,

operon

needs to be turned on. Allolactose (inducer) binds to repressor, changes its shape making it drop off leaving RNA polymerase to start transcriptionSlide12

Inducible enzymes

Usually function in catabolic pathwaysLactose broken down into glucose and galactoselac operon to produce

B

-

galactosidaseRepressible enzymesUsually function in anabolic pathwaysTryptophan being producedtrp operon to produce tryptophanSlide13

Regulation of both the trp

and lac operonsInvolves the negative control of genes, because the operons are switched off by the active form of the repressor proteinSlide14

Positive Gene Regulation

If Lactose is present and glucose is lacking, E. coli will break down lactose for energy but how does it sense that glucose is lacking?

It senses this through an

allosteric

regulatory protein called Catabolite Activator Protein (CAP) that binds to a small molecule called

cyclic AMP (

cAMP

)

CAP

acts

as an activator to bind to DNA to stimulate transcription of a gene (such as the lac operon)Think of it like cAMP pushing CAP to make RNA polymerase go faster to make the enzymes to break lactose down to glucoseSlide15

Promoter

Lactose present

,

glucose scarce

(

cAMP

level high

):

abundant

lac

mRNA synthesized

.If glucose is scarce, the high level of cAMP activates CAP, and the lac operon

produces large amounts of mRNA for the lactose pathway.

(a)

CAP-binding site

Operator

RNApolymerasecan bindand transcribe

InactiveCAP

Active

CAP

cAMPDNA

Inactive

lacrepressor

lacl

lacZIn E. coli, when glucose, (a preferred food source), is scarce, cAMP (cyclic AMP) builds upthe lac operon is activated by the binding of a regulatory protein, catabolite activator protein

(CAP) activated by the binding of cAMP to CAP changing its conformation. Thus allowing transcriptioncAMP is the green light that tells CAP to floor it!Slide16
Slide17

When

glucose levels in an E. coli cell increases (& lactose levels are low) there is a decrease in cAMP. CAP

detaches from the lac operon due to the release of

cAMP

to CAP changing its conformation, & turning it off Animation

Figure 18.23b

(b)

Lactose present

,

glucose present

(

cAMP

level low): little lac mRNA synthesized. When glucose is present, cAMP is scarce, and CAP is unable to stimulate transcription.

Inactive lacrepressor

InactiveCAP

DNA

RNApolymerasecan’t bind

Operator

lacl

lacZ

CAP-binding site

PromoterSlide18

Eukaryotic gene expression is regulated at many stages

All organisms must regulate which genes are expressed at any given time. Which ones are turned on or off.

In

multicellular

organisms regulation of gene expression is essential for cell specialization~20% of protein coding genes are being expressed at any time in human cellsSlide19

Differential Gene Expression

Almost all the cells in an organism are genetically identical with the exception of gametesDifferences between cell types result from differential gene expression, the expression of different genes by cells with the same genome

RBC from an epithelial cell to a nerve cells

Abnormalities in gene expression can lead to diseases including

cancerGene expression is regulated at many stagesSlide20

Signal

NUCLEUS

Chromatin

Chromatin modification:

DNA unpacking involvinghistone acetylation andDNA demethylationDNAGene

Gene available

for transcription

RNA

Exon

Primary transcript

TranscriptionIntron

RNA processingCapTailmRNA in nucleusTransport to cytoplasmCYTOPLASM

mRNA in cytoplasmTranslation

Degradationof mRNAPolypeptideProtein processing, suchas cleavage and chemical modificationActive proteinDegradationof protein

Transport to cellulardestinationCellular function (suchas enzymatic activity,structural support)Stages in gene expression that can be regulated in eukaryotic cells.Slide21

Signal

NUCLEUS

Chromatin

Chromatin modification:

DNA unpacking involvinghistone acetylation andDNA demethylationDNA

Gene

Gene available

for transcription

RNA

Exon

Primary transcriptTranscriptionIntron

RNA processingCapTailmRNA in nucleusTransport to cytoplasm

CYTOPLASMSlide22

Figure 18.6b

CYTOPLASM

mRNA in cytoplasm

Translation

Degradationof mRNAPolypeptideProtein processing, such

as cleavage and

chemical modification

Active protein

Degradation

of protein

Transport to cellulardestinationCellular function (suchas enzymatic activity,structural support)Slide23

Regulation of Chromatin Structure

The genes within highly packed heterochromatin (DNA + proteins) are usually not expressedEuchromatin

is found in eukaryotes. Less compacted DNA

Chemical modifications to

histones and DNA of chromatin influence both chromatin structure and gene expressionAcetylation- allows transcriptionMethylation – blocks transcriptionPhosphorylation – promotes transcriptionSlide24

Histone

ModificationsFor the Transcription of a gene on DNA to occur, histones

must be modified to uncoil the DNA to allow for transcription

In

histone acetylation, acetyl groups (-COCH3) are attached to positively charged lysines in histone tailsHistone tails no longer will bind to adjacent nucleosomesThis loosens chromatin structure, thereby promotes the initiation of transcriptionHistone

acetylation

enzymes may

promote the initiation of transcription

.

Kosigrin animationSlide25

Amino acids

availablefor chemicalmodification

Histone

tails

DNA double helixNucleosome(end view)(a) Histone tails protrude outward from a nucleosome

Unacetylated histones

Acetylated histones

(b)

Acetylation of histone tails promotes loose chromatin

structure that permits transcriptionSlide26

The

addition of methyl groups – CH3 to Cystosine (

methylation

) to histone tails promotes

condensing of chromatin.So this turns off genesThe addition of phosphate groups (phosphorylation) next to a methylated amino acid can loosen chromatinTurns on genesSlide27

DNA Methylation

DNA methylation, the addition of methyl groups to certain bases in DNA, is associated with reduced transc

ription

in some speciesDNA methylation can cause long-term inactivation of genes in cellular differentiationSuch as in X inactivation when the X chromosomes become methylatedUnexpressed genes are more heavily methylated than active genes.Causes suppression of cancer suppressor genes (p53)Slide28

Epigenetic Inheritance

Although the chromatin modifications do not alter DNA sequence, they may be passed to future generations of cellsThe inheritance of traits transmitted by mechanisms not directly involving the nucleotide sequence is called

epigenetic inheritance

Caused by DNA

methylation and therefore certain enzymes are not produced which may be necessary for cell functionMay be the cause of some cancersGhosts in Your GenesEpigenetics videoSlide29
Slide30

Control & Organization of a Typical Eukaryotic Gene

Chromatin-modifying enzymes provide initial control of gene expression by making a region of DNA either more or less able to bind the transcription machineryAssociated with most eukaryotic genes are multiple

control elements

, segments of noncoding DNA that serve as binding sites for transcription factors that help regulate transcriptionControl elements and the transcription factors they bind to are critical to the precise regulation of gene expression in different cell typesSlide31

Enhancer

(distal control

elements)

DNA

UpstreamPromoter

Proximal

control

elements

Transcription

start site

ExonIntronExon

ExonIntronPoly-AsignalsequenceTranscriptionterminationregion

Downstream

A eukaryotic gene and its transcript.Slide32

Enhancer

(distal controlelements)

DNA

Upstream

PromoterProximalcontrolelements

Transcription

start site

Exon

Intron

Exon

Exon

IntronPoly-AsignalsequenceTranscriptionterminationregionDownstreamPoly-Asignal

ExonIntronExonExonIntronTranscription

Cleaved3 end ofprimarytranscript5Primary RNAtranscript(pre-mRNA)

A

eukaryotic gene and its transcript.Slide33

Enhancer

(distal controlelements)

DNA

Upstream

PromoterProximalcontrolelements

Transcription

start site

Exon

Intron

Exon

Exon

IntronPoly-AsignalsequenceTranscriptionterminationregionDownstreamPoly-Asignal

ExonIntronExonExonIntron

TranscriptionCleaved3 end ofprimarytranscript5Primary RNAtranscript(pre-mRNA)

Intron RNARNA processingmRNACoding segment5 Cap

5 UTRStartcodonStopcodon3 UTR3

Poly-AtailPPPGAAA  AAA

A

eukaryotic gene and its transcript.Slide34

The Roles of Transcription Factors

To initiate transcription, eukaryotic RNA polymerase requires the assistance of proteins called transcription factorsGeneral 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 factorsSlide35

Proximal

control elements are located close to the promoterDistal control elements, groupings of which are called enhancers, may be far away from a gene or even located in an intron

These control elements could be thousands of base pairs away from the gene on the DNA strand

Enhancers

and Specific Transcription FactorsSlide36

An

activator is a protein that binds to an enhancer on the control factor and

stimulates transcription of a gene.

Think of it like the key that gets transcription going

Activators have two domains, one that binds to DNA and a second that activates transcriptionBound activators facilitate a sequence of protein-protein interactions that result in transcription of a given geneMcGraw Hill animationSlide37

Some transcription factors function

as repressors, inhibiting expression of a particular gene by a variety of methodsSome activators and repressors act indirectly by influencing chromatin structure to promote or silence transcriptionSlide38

Activators

DNA

Enhancer

Distal

controlelementPromoter

Gene

TATA box

A model for the action of enhancers and transcription activatorsSlide39

Activators

DNA

Enhancer

Distal control

elementPromoterGene

TATA box

General

transcription

factors

DNA-

bendingproteinGroup of mediator proteins

A model for the action of enhancers and transcription activatorsSlide40

Activators

DNA

Enhancer

Distal control

elementPromoterGene

TATA box

General

transcription

factors

DNA-

bendingproteinGroup of mediator proteinsRNApolymerase IIRNApolymerase

IIRNA synthesisTranscriptioninitiation complex

AnimationA model for the action of enhancers and transcription activatorsSlide41

A particular combination of

control elements can activate transcription only when the appropriate activator proteins are presentand therefore not allow the transcription of other genesLike combinations on a lock. Same lock, different combinations. Or passwords on your computer. Same computer but different passwords for different people

Combinatorial

Control of Gene ActivationSlide42

Control

elements

Enhancer

Promoter

Albumin geneCrystallingene

LIVER CELL

NUCLEUS

Available

activators

Albumin gene

expressedCrystallin genenot expressed

(a) Liver cellLENS CELLNUCLEUSAvailableactivatorsAlbumin genenot expressedCrystallin

geneexpressed(b) Lens cell

Cell type–specific transcriptionSlide43

Control

elements

Enhancer

Promoter

Albumin geneCrystallingene

LIVER CELL

NUCLEUS

Available

activators

Albumin gene

expressedCrystallin genenot expressed(a) Liver cell

Cell type–specific transcriptionSlide44

Control

elements

Enhancer

Promoter

Albumin geneCrystallingeneLENS CELL

NUCLEUS

Available

activators

Albumin gene

not expressed

Crystallin geneexpressed(b) Lens cell

Cell type–specific transcriptionSlide45

Coordinately Controlled Genes in Eukaryotes

Unlike the genes of a prokaryotic operon, each of the co-expressed eukaryotic genes has a promoter and control elementsThese 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 genesSlide46

Mechanisms of Post-Transcriptional Regulation

Transcription alone does not account for gene expressionRegulatory mechanisms can operate at various stages after transcriptionSuch mechanisms allow a cell to fine-tune gene expression rapidly in response to environmental changesSlide47

RNA Processing

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

intronsSlide48

Exons

DNA

Troponin T gene

Primary

RNAtranscriptRNA splicingor

mRNA

1

1

1

1

2

222333

44455

55Slide49

Initiation of Translation

The initiation of translation of selected mRNAs can be blocked by regulatory proteins

that bind to sequences or structures of the mRNA

Alternatively,

translation of all mRNAs in a cell may be regulated simultaneouslyFor example, translation initiation factors are simultaneously activated in an egg following fertilizationSlide50

Protein Processing and Degradation

After translation, various types of protein processing, including cleavage (such as with pro-insulin which needs to be altered to become active insulin) the addition of chemical groups, are subject to control (phosphorylation)Chemical modifications by the addition/removal of phosphate groups.Functional length (of time) is regulator so that the protein is only present while it is needed (such as with

cyclin

).

To mark a protein for destruction, ubiquitin is added to it.Slide51

Chromatin modification

• Genes in highly compacted

chromatin are generally not

transcribed.

• Histone acetylation seemsto loosen chromatin structure,enhancing transcription.• DNA

methylation

generally

reduces transcription.

• 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 forliver-specific genesEnhancer forlens-specific genesTranscriptionRNA processing• Alternative RNA splicing:

Primary RNAtranscriptmRNAorChromatin modificationTranscriptionRNA processing

mRNAdegradationTranslationProtein processingand degradationSlide52

mRNA degradation

• Each mRNA has a characteristic life span,

determined in part by

sequences in the 5

 and3 UTRs.• 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

TranslationProtein processingand degradation