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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 expression.

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chapter 15 regulation of gene expression

Chapter 15: Regulation of Gene Expression

Anablepanablepwith 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 expression

slide2

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. colilives in the human colon & can fine-tune its metabolism to the changing environment and food sources
  • If tryptohan is lacking, the cell responds by activating a gene that starts a metabolic pathway to produce tryptophanfrom another food source that is present.
  • If tryptophan is plentiful, the bacteria stop making tryptophan to conserve energy.
slide3

(a) Regulation of enzyme activity

(b) Regulation of enzyme production

Feedback

inhibition

Precursor

Enzyme 1

Gene 1

Regulation

of gene

expression

Gene 2

Enzyme 2

Gene 3

Enzyme 3

Enzyme 4

Gene 4

Enzyme 5

Gene 5

Tryptophan

Figure 18.20a, b

  • 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 response
operons the basic concept
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”switchwhich is a segment of DNA that lies betweenpromoter and enzyme-coding genes
    • A promoter – 1 for the entire operon
    • Genes for metabolic enzymes Animation
animation

trp operon

Promoter

Promoter

Genes of operon

RNA polymerase

Start codon Stop codon

trpR

trpD

trpC

trpB

trpE

trpA

DNA

Operator

Regulatory

gene

3

mRNA 5

mRNA

5

C

E

D

B

A

Polypeptides that make up

enzymes for tryptophan synthesis

Inactiverepressor

Protein

  • Tryptophan absent, repressor inactive, operonon.
  • RNA polymerase attaches to the DNA at the promoter and transcribes the operon’s genes.

Figure 18.21a

Animation
  • The Trpoperon
    • Is usually turned “On” (with no repressor protein stopping transcription)
    • Can be switched off by a protein called a repressor protein
  • trpR is a regulatory gene which is continually expressed in small amounts. It produces the repressor protein that binds to Operator to prevent transcription
slide7

DNA

No RNA made

mRNA

Active

repressor

Protein

Tryptophan

(corepressor)

Tryptophan present, repressor active, operonoff.

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

(b)

Operator

trpR

Regulatory

gene

RNA polymerase

Inactiverepressor

repressible and inducible operons two types of negative gene regulation
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 toallostericallyto regulatory protein
    • Binding of a specific repressor protein to the operator shuts off transcription
    • Trpoperon
  • 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 transcription
slide9

Promoter

Regulatorygene

Operator

DNA

lacl

lacZ

NoRNAmade

3

RNApolymerase

mRNA

5

Activerepressor

Protein

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)

Figure 18.22a

  • The lac operon: regulated synthesis of Inducible enzymes.
  • Regulates the catabolism of lactose to galactose and glucose
  • Needs an inducer to remove repressor and start transcription
  • animation
slide10

lac operon

DNA

lacl

lacz

lacY

lacA

RNApolymerase

3

mRNA 5

mRNA 5'

mRNA

5

-Galactosidase

Permease

Transacetylase

Protein

Inactiverepressor

Allolactose(inducer)

When Lactose is present, the repressor is inactive, operonis on & β –galactosidase is produced.

Allolactose, an isomer of lactose, acts as the inducer by derepressesthe operon by inactivating the repressor.

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

(b)

slide11

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 transcription

slide12

Inducible enzymes

    • Usually function in catabolic pathways
    • Lactose broken down into glucose and galactose
    • lacoperon to produce B - galactosidase
  • Repressible enzymes
    • Usually function in anabolic pathways
    • Tryptophan being produced
    • trpoperon to produce tryptophan
slide13

Regulation of both the trp and lac operons

    • Involves the negative control of genes, because the operons are switched off by the active form of the repressor protein
positive gene regulation
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 CataboliteActivator 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 lacoperon)
  • Think of it like cAMP pushing CAP to make RNA polymerase go faster to make the enzymes to break lactose down to glucose
slide15

Operator

RNA

polymerase

can bindand transcribe

Promoter

DNA

lacl

lacZ

CAP-binding site

cAMP

ActiveCAP

Inactive lac

repressor

InactiveCAP

(a)

Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized.If glucose is scarce, the high level of cAMP activates CAP, and the lacoperon produces large amounts of mRNA for the lactose pathway.

  • In E. coli, when glucose, (a preferred food source), is scarce, cAMP (cyclic AMP) builds up
    • the 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 transcription
    • cAMP is the green light that tells CAP to floor it!
slide17

Promoter

Operator

DNA

lacl

lacZ

CAP-binding site

RNA

polymerase

can’t bind

InactiveCAP

Inactive lac

repressor

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

(b)

Figure 18.23b

  • When glucose levels in an E. coli cell increases(& lactose levels are low) there is a decrease in cAMP. CAPdetaches from the lacoperon due to the release of cAMP to CAP changing its conformation, & turning it off
  • Animation
eukaryotic gene expression is regulated at many stages
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 cells
differential gene expression
Differential Gene Expression
  • Almost all the cells in an organism are genetically identical with the exception of gametes
  • Differences 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 cancer
  • Gene expression is regulated at many stages
slide20

Signal

NUCLEUS

Stages in gene expression that can be regulated in eukaryotic cells.

Chromatin

Chromatin modification:DNA unpacking involvinghistone acetylation andDNA demethylation

DNA

Gene availablefor transcription

Gene

Transcription

Exon

RNA

Primary transcript

Intron

RNA processing

Tail

mRNA in nucleus

Cap

Transport to cytoplasm

CYTOPLASM

mRNA in cytoplasm

Translation

Degradationof mRNA

Polypeptide

Protein processing, suchas cleavage and chemical modification

Active protein

Degradationof protein

Transport to cellulardestination

Cellular function (suchas enzymatic activity,structural support)

slide21

Signal

NUCLEUS

Chromatin

Chromatin modification:DNA unpacking involvinghistone acetylation andDNA demethylation

DNA

Gene availablefor transcription

Gene

Transcription

Exon

RNA

Primary transcript

Intron

RNA processing

Tail

mRNA in nucleus

Cap

Transport to cytoplasm

CYTOPLASM

figure 18 6b

CYTOPLASM

Figure 18.6b

mRNA in cytoplasm

Translation

Degradationof mRNA

Polypeptide

Protein processing, suchas cleavage and chemical modification

Active protein

Degradationof protein

Transport to cellulardestination

Cellular function (suchas enzymatic activity,structural support)

regulation of chromatin structure
Regulation of Chromatin Structure
  • The genes within highly packed heterochromatin (DNA + proteins) are usually not expressed
    • Euchromatin is found in eukaryotes. Less compacted DNA
  • Chemical modifications to histonesand DNA of chromatin influence both chromatin structure and gene expression
      • Acetylation- allows transcription
      • Methylation – blocks transcription
      • Phosphorylation – promotes

transcription

histone modifications
Histone Modifications

For the Transcription of a gene on DNA to occur, histones must be modified to uncoil the DNA to allow for transcription

  • In histoneacetylation, acetyl groups (-COCH3) are attached to positively charged lysinesin histone tails
    • Histone tails no longer will bind to adjacent nucleosomes
    • This loosens chromatin structure, thereby promotes the initiation of transcription
    • Histoneacetylation enzymes may

promote the initiation of transcription.

Kosigrin animation

Mc Graw Hill

slide25

Histone tails

DNA double helix

Amino acidsavailablefor chemicalmodification

Nucleosome(end view)

(a) Histone tails protrude outward from a nucleosome

Acetylated histones

Unacetylated histones

(b)

Acetylation of histone tails promotes loose chromatinstructure that permits transcription

slide26

The addition of methyl groups – CH3 to Cystosine(methylation) to histone tails promotes condensing of chromatin.

    • So this turns off genes
  • The addition of phosphate groups (phosphorylation) next to a methylated amino acid canloosen chromatin
    • Turns on genes
dna methylation
DNA Methylation
  • DNA methylation, the addition of methyl groups to certain bases in DNA, is associated with reduced transcriptionin some species
  • DNA methylation can cause long-term inactivation of genes in cellular differentiation
    • Such as in X inactivation when the X chromosomes become methylated
    • Unexpressed genes are more heavily methylated than active genes.
epigenetic inheritance
Epigenetic Inheritance
  • Although the chromatin modifications 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
  • Caused by DNA methylation and therefore certain enzymes are not produced which may be necessary for cell function
  • May be the cause of some cancers
  • Ghosts in Your Genes
  • Epigenetics video
control organization of a typical eukaryotic gene
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 machinery
  • Associated with most eukaryotic genes are multiplecontrol elements, segments of noncoding DNA that serve as binding sites for transcription factorsthat help regulate transcription
  • Control elements and the transcription factors they bind are critical to the precise regulation of gene expression in different cell types
slide31

A eukaryotic gene and its transcript.

Proximalcontrolelements

Enhancer(distal controlelements)

Poly-Asignalsequence

Transcriptionterminationregion

Transcriptionstart site

Exon

Exon

Intron

Intron

Exon

DNA

Upstream

Downstream

Promoter

slide32

A eukaryotic gene and its transcript.

Enhancer(distal controlelements)

Poly-Asignalsequence

Proximalcontrolelements

Transcriptionterminationregion

Transcriptionstart site

Exon

Intron

Intron

Exon

Exon

DNA

Upstream

Downstream

Promoter

Poly-Asignal

Transcription

Exon

Intron

Intron

Exon

Exon

Primary RNAtranscript(pre-mRNA)

Cleaved3 end ofprimarytranscript

5

slide33

A eukaryotic gene and its transcript.

Enhancer(distal controlelements)

Proximalcontrolelements

Poly-Asignalsequence

Transcriptionterminationregion

Transcriptionstart site

Exon

Intron

Intron

Exon

Exon

DNA

Upstream

Downstream

Promoter

Poly-Asignal

Transcription

Exon

Intron

Intron

Exon

Exon

Primary RNAtranscript(pre-mRNA)

Cleaved3 end ofprimarytranscript

5

RNA processing

Intron RNA

Coding segment

mRNA

3

G

P

P

P

AAA AAA

Startcodon

Stopcodon

Poly-Atail

5 UTR

5 Cap

3 UTR

the roles of transcription factors
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
slide35

Enhancers and 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
    • These control elements could be thousands of base pairs away from the gene on the DNA strand
slide36
An activator is a protein that binds to an enhancer on the control factor and stimulates transcription of a gene
  • Activators have two domains, one that binds to DNA and a second that activates transcription
  • Bound activators facilitate a sequence of protein-protein interactions that result in transcription of a given gene

McGraw Hill animation

slide37
Some transcription factors function as repressors, inhibiting expression of a particular geneby a variety of methods
  • Some activators and repressors act indirectly by influencing chromatin structure to promote or silence transcription
slide38

Promoter

Activators

Gene

DNA

Distal controlelement

Enhancer

TATA box

A model for the action of enhancers and transcription activators

slide39

Promoter

Activators

Gene

DNA

Distal controlelement

TATA box

Enhancer

Generaltranscriptionfactors

DNA-bendingprotein

Group of mediator proteins

A model for the action of enhancers and transcription activators

slide40

Promoter

Activators

Gene

DNA

Distal controlelement

TATA box

Enhancer

Generaltranscriptionfactors

DNA-bendingprotein

Group of mediator proteins

RNApolymerase II

Animation

RNApolymerase II

Transcriptioninitiation complex

RNA synthesis

A model for the action of enhancers and transcription activators

slide41

Enhancer

Promoter

Controlelements

Albumin gene

Cell type–specific transcription

Crystallingene

LENS CELLNUCLEUS

LIVER CELLNUCLEUS

Availableactivators

Availableactivators

Albumin genenot expressed

Albumin geneexpressed

Crystallin genenot expressed

Crystallin geneexpressed

(a) Liver cell

(b) Lens cell

slide42

Enhancer

Promoter

LIVER CELLNUCLEUS

Controlelements

Albumin gene

Availableactivators

Crystallingene

Albumin geneexpressed

Crystallin genenot expressed

(a) Liver cell

Cell type–specific transcription

slide43

Enhancer

Promoter

LENS CELLNUCLEUS

Controlelements

Albumin gene

Availableactivators

Crystallingene

Albumin genenot expressed

Crystallin geneexpressed

(b) Lens cell

Cell type–specific transcription

coordinately controlled genes in eukaryotes
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
mechanisms of post transcriptional regulation
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
rna processing
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 introns
slide47

Exons

DNA

4

1

3

5

2

Troponin T gene

PrimaryRNAtranscript

3

5

2

4

1

RNA splicing

or

mRNA

3

5

5

2

2

4

1

1

initiation of translation
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 simultaneously
  • For example, translation initiation factors are simultaneously activated in an egg following fertilization
protein processing and degradation
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) and the addition of chemical groups, are subject to control (phosporylation)
  • Chemical modifications by the addition/removal of phosphate groups.
  • Functional length 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.
slide50

Chromatin modification

Transcription

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

• Genes in highly compactedchromatin are generally nottranscribed.

• Histoneacetylation seemsto loosen chromatin structure,enhancing transcription.

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

• DNA methylation generallyreduces transcription.

• Coordinate regulation:

Enhancer forlens-specific genes

Enhancer forliver-specific genes

Chromatin modification

Transcription

RNA processing

• Alternative RNA splicing:

RNA processing

Primary RNAtranscript

Translation

mRNAdegradation

mRNA

or

Protein processingand degradation

slide51

Chromatin modification

Transcription

RNA processing

Translation

mRNAdegradation

Protein processingand degradation

Translation

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

mRNA degradation

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

Protein processing and degradation

• Protein processing anddegradation by proteasomesare subject to regulation.