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

• 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 sources– If 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.

Figure 18.20a, b

(a) Regulation of enzyme activity

Enzyme 1

Enzyme 2

Enzyme 3

Enzyme 4

Enzyme 5

Regulationof geneexpression

Feedbackinhibition

Tryptophan

Precursor

(b) Regulation of enzyme production

Gene 2

Gene 1

Gene 3

Gene 4

Gene 5

–

–

• This metabolic control occurs on two levels1. 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 1Short term response

2. Regulating the genes encoding the metabolic enzymes• Tryptophan can shut down the transcription of a gene is a longer term response

Operons: The Basic ConceptIn bacteria, genes are often clustered into groups of

genes (such as the 5 for tryptophan) called operons, composed of:1. An operator, an “on-off” switch which is a segment of DNA that lies

between promoter and enzyme-coding genes2. A promoter – 1 for the entire operon

3. Genes for metabolic enzymes Animation

Animation

• The Trp operon– 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

Figure 18.21a

(a) 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

Protein

Operator

Polypeptides that make upenzymes for tryptophan synthesis

Promoter

Regulatorygene

RNA polymerase

Start codon Stop codon

Promoter

trp operon

5

3mRNA 5

trpDtrpE trpC trpB trpAtrpRDNA

mRNA

E D C B A

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

Operator

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 protein

– Binding of a specific repressor protein to the operator shuts off transcription

– Trp 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 transcription

Figure 18.22a

DNA

mRNA

ProteinActiverepressor

RNApolymerase

NoRNAmade

lacZlacl

Regulatorygene 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 glucose• Needs an inducer to remove repressor and start transcription

• animation

mRNA 5'

DNA

mRNA

Protein

Allolactose(inducer)

Inactiverepressor

lacl lacz lacY lacA

RNApolymerase

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 operon

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

b. 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

• Inducible enzymes– Usually function in catabolic pathways– Lactose broken down into glucose and galactose– lac operon to produce B - galactosidase

• Repressible enzymes– Usually function in anabolic pathways– Tryptophan being produced– trp operon to produce tryptophan

• 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• 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 glucose

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 OperatorRNApolymerasecan bindand transcribe

InactiveCAP

ActiveCAP

cAMP

DNA

Inactive lacrepressor

lacl lacZ

• 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!

• 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

Promoter

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

• 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

Signal

NUCLEUSChromatin

Chromatin modification:DNA unpacking involvinghistone acetylation andDNA demethylation

DNA

Gene

Gene availablefor transcription

RNA ExonPrimary transcript

Transcription

IntronRNA processing

Cap

TailmRNA in nucleus

Transport to cytoplasm

CYTOPLASMmRNA in cytoplasm

TranslationDegradationof mRNA

Polypeptide

Protein processing, suchas cleavage and chemical modification

Active proteinDegradationof protein

Transport to cellulardestination

Cellular function (suchas enzymatic activity,structural support)

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

Signal

NUCLEUSChromatin

Chromatin modification:DNA unpacking involvinghistone acetylation andDNA demethylation

DNA

Gene

Gene availablefor transcription

RNA ExonPrimary transcript

Transcription

IntronRNA processing

Cap

TailmRNA in nucleus

Transport to cytoplasm

CYTOPLASM

Figure 18.6bCYTOPLASM

mRNA in cytoplasm

TranslationDegradationof mRNA

Polypeptide

Protein processing, suchas cleavage and chemical modification

Active proteinDegradationof protein

Transport to cellulardestination

Cellular function (suchas enzymatic activity,structural support)

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 histones and DNA of

chromatin influence both chromatin structure and gene expression

• Acetylation- allows transcription• Methylation – blocks transcription• Phosphorylation – promotes transcription

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 histone acetylation, acetyl groups (-COCH3) are attached to positively charged lysines in histone tails– Histone tails no longer will bind to adjacent nucleosomes– This loosens chromatin structure, thereby promotes the

initiation of transcription– Histone acetylation enzymes may promote the initiation of transcription.Kosigrin animation

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 chromatin

structure that permits transcription

• 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 can loosen chromatin– Turns on genes

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– Such as in X inactivation when the X chromosomes become

methylated– Unexpressed genes are more heavily methylated than

active genes.– Causes suppression of cancer suppressor genes (p53)

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

• 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 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 to are critical to the precise regulation of gene expression in different cell types

Enhancer(distal controlelements)

DNA

Upstream Promoter

Proximalcontrolelements

Transcriptionstart site

Exon Intron Exon ExonIntron

Poly-Asignalsequence

Transcriptionterminationregion

Downstream

A eukaryotic gene and its transcript.

Enhancer(distal controlelements)

DNA

Upstream Promoter

Proximalcontrolelements

Transcriptionstart site

Exon Intron Exon ExonIntron

Poly-Asignalsequence

Transcriptionterminationregion

DownstreamPoly-Asignal

Exon Intron Exon ExonIntron

Transcription

Cleaved3 end ofprimarytranscript

5Primary RNAtranscript(pre-mRNA)

A eukaryotic gene and its transcript.

Enhancer(distal controlelements)

DNA

Upstream Promoter

Proximalcontrolelements

Transcriptionstart site

Exon Intron Exon ExonIntron

Poly-Asignalsequence

Transcriptionterminationregion

DownstreamPoly-Asignal

Exon Intron Exon ExonIntron

Transcription

Cleaved3 end ofprimarytranscript

5Primary RNAtranscript(pre-mRNA)

Intron RNA

RNA processing

mRNA

Coding segment

5 Cap 5 UTRStartcodon

Stopcodon 3 UTR

3

Poly-Atail

PPPG AAA AAA

A eukaryotic gene and its transcript.

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– These control elements could be thousands of base pairs

away from the gene on the DNA strand

Enhancers and Specific Transcription Factors

• 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 transcription

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

McGraw Hill animation

• 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

Enhancer Distal controlelement

PromoterGene

TATA box

A model for the action of enhancers and transcription activators

ActivatorsDNA

Enhancer Distal controlelement

PromoterGene

TATA boxGeneraltranscriptionfactors

DNA-bendingprotein

Group of mediator proteins

A model for the action of enhancers and transcription activators

ActivatorsDNA

Enhancer Distal controlelement

PromoterGene

TATA boxGeneraltranscriptionfactors

DNA-bendingprotein

Group of mediator proteins

RNApolymerase II

RNApolymerase II

RNA synthesisTranscriptioninitiation complex

Animation

A model for the action of enhancers and transcription activators

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

Cell type–specific transcription

Controlelements

Enhancer Promoter

Albumin gene

Crystallingene

LIVER CELLNUCLEUS

Availableactivators

Albumin geneexpressed

Crystallin genenot expressed

(a) Liver cellCell type–specific transcription

Controlelements

Enhancer Promoter

Albumin gene

Crystallingene

LENS CELLNUCLEUS

Availableactivators

Albumin genenot expressed

Crystallin geneexpressed

(b) Lens cellCell type–specific transcription

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

• 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• 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

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

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

• 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.

Chromatin modification

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

• DNA methylation generallyreduces 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 genes

Enhancer forlens-specific genes

Transcription

RNA processing

• Alternative RNA splicing:

Primary RNAtranscript

mRNA or

Chromatin modification

Transcription

RNA processing

mRNAdegradation

Translation

Protein processingand degradation

mRNA degradation

• Each mRNA has a characteristic life span,determined in part bysequences 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

Translation

Protein processingand degradation