Chapter 18

Regulation of Gene Expression

Overview: Conducting the Genetic Orchestra

• 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

Wild type Mutant

Eye

AntennaLeg

Bacteria regulate their gene expression

• Feedback mechanisms allow control over metabolism so that cells produce only the products needed at that time (with some limitations)

• This metabolic control occurs on two levels:

1. adjusting activity of metabolic enzymes

2. regulating genes that encode metabolic enzymes

Regulationof geneexpression

trpE gene

trpD gene

trpC gene

trpB gene

trpA gene

(b) Regulation of enzyme production

(a) Regulation of enzyme activity

Enzyme 1

Enzyme 2

Enzyme 3

Tryptophan

PrecursorFeedbackinhibition

In the pathway for tryptophan synthesis, an abundance of tryptophan can both (a)inhibit the activity of the first enzyme in the pathway (feedback inhibition), a rapid response, and(b)repress expression of the genes for all the enzymes needed for the pathway, a longer-term response.

Regulation of a Metabolic Pathway

• In bacteria, genes are often clustered into operons, composed of:

1. an operator, an “on-off” switch

2. a promoter

3. genes for metabolic enzymes

• An operon can be switched off by a protein called a repressor– binds only to the operator

Promoter Promoter

DNA trpR

Regulatorygene RNA

polymerase

mRNA

3

5

Protein Repressor

Tryptophan absent, repressor inactive, operon on

mRNA 5

trpE trpD trpC trpB trpA

OperatorStart codonStop codon

trp operon

Genes of operon (5)

E

Polypeptides (5) that make upenzymes for tryptophan synthesis

D C B A

operonON

no tryptophan

repressorinactive

(inactive)

Operons

• 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

– repressible enzymes usually function in anabolic pathways• cells suspend production of an end product that is not needed

DNA

Protein

Tryptophan(corepressor)

Tryptophan present, repressor active, operon off

mRNA

Activerepressor

DNA

Protein

Tryptophan(corepressor)

mRNA

Activerepressor

No RNA made

corepressor- a small molecule that cooperates with a repressor to switch an operon off

The trp operonoperon

OFFtryptophan repressor

active

Operons

• 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

– repressible enzymes usually function in anabolic pathways• cells suspend production of an end product that is not needed

• An inducible operon-is one that is usually off; a molecule, an inducer, inactivates the repressor & turns on transcription

– the classic example of an inducible operon is the lac operon

– inducible enzymes usually function in catabolic pathways• cells only produce enzymes when there’s a nutrient that needs to be broken down

DNA lacl

Regulatorygene

mRNA

5

3

RNApolymerase

ProteinActiverepressor

NoRNAmade

lacZ

Promoter

Operator

Lactose absent, repressor active, operon off

The lac operon

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

operonOFF

no lactose

repressoractive

DNA lacl

mRNA5

3

lac operon

Lactose present, repressor inactive, operon on

lacZ lacY lacA

RNApolymerase

mRNA 5

Protein

Allolactose(inducer)

Inactiverepressor

-Galactosidase Permease Transacetylase

Allolactose, an isomer of lactose, derepresses the operon by inactivating the repressor. In this way, the enzymes for lactose utilization are induced.

3 genes

E.Coli uses 3 enzymes to take up and metabolize lactose

β-galactosidase: hydrolyzes lactose to glucose and galactosepermease: transports lactose into the celltransacetylase: function in lactose metabolism is still unclear

operonON

lactose repressorinactive

inducer- a small molecule that inactivates the repressor

Gene Regulation

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

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

• The lac operon is under dual control:– negative control by the lac repressor– positive control by CAP

DNA

cAMP

lacl

CAP-binding site

Promoter

ActiveCAP

InactiveCAP

RNApolymerasecan bindand transcribe

Operator

lacZ

Inactive lacrepressor

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

• When glucose (a preferred food source of E. coli ) is scarce, the lac operon is activated by the binding of CAP

Positive Control

glucose

cAMP

DNA lacl

CAP-binding site

Promoter

RNApolymerasecan’t bind

Operator

lacZ

Inactive lacrepressor

InactiveCAP

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

When glucose levels increase, CAP detaches from the lac operon, turning it off

Promoter

Genes

Genes not expressed

Active repressor:no inducer present

Inactive repressor:inducer bound

Genes expressed

Operator

Promoter

Genes

Genes not expressed

Inactive repressor:no corepressor present Corepressor

Active repressor:corepressor bound

Genes expressed

Operator

REVIEWRepressible Operon

Inducible Operon

Eukaryotic Genomes

• Two features of eukaryotic genomes are a major information-processing challenge:

1. the typical eukaryotic genome is much larger than that of a prokaryotic cell

2. cell specialization limits the expression of many genes to specific cells

The DNA-protein complex, called chromatin, is ordered into higher structural levels than the DNA-protein complex in prokaryotes

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

• Errors in gene expression can lead to diseases including cancer

Signal

NUCLEUSChromatin

Chromatin modification:DNA unpacking involvinghistone acetylation andDNA demethylation

DNA

Gene

Gene availablefor transcription

RNA ExonPrimary transcript

Transcription

Intron

RNA processing

Cap

Tail

mRNA in nucleus

Transport to cytoplasm

CYTOPLASM

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)

Gene Expression

• Gene expression is regulated at many stages

#1

#2

#3

#4

#6

***** most important control point

#5

#6

• Genes within highly packed heterochromatin (highly condensed areas) are usually not expressed

• Chemical modifications to histones and DNA of chromatin influence both chromatin structure and gene expression

• histone acetylation-

acetyl groups (-COCH3) are attached to positively charged lysines in histone tails

• This process seems to loosen chromatin structure, thereby promoting the initiation of transcription

Histonetails

Amino acidsavailablefor chemicalmodification

DNAdouble helix

Histone tails protrude outward from a nucleosome

Acetylation of histone tails promotes loose chromatinstructure that permits transcription

Unacetylated histones Acetylated histones

Histones#1

#1

#2

#3

#4

#6

#5

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 turns off either the maternal or paternal alleles of certain genes at the start of development

#1

Summary- Chromatin Modifications

• 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

• 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

#1

Eukaryotic Gene• Associated with most eukaryotic genes are multiple control elements,

segments of noncoding DNA that help regulate transcription by binding certain proteins

– Control elements & the proteins they bind are critical to the precise regulation of gene expression in different cell types

Enhancer(distal control elements)

Proximal control elements

Upstream

DNA

Promoter

Exon Intron Exon Intron Exon

DownstreamTranscription

Poly-A signalsequence

Terminationregion

Intron Exon Intron Exon

RNA processing:Cap and tail added;introns excised andexons spliced together

Poly-A signal

Cleaved 3 endof primarytranscript

3

Poly-Atail

3 UTR(untranslated

region)

5 UTR(untranslated

region)

Startcodon

Stopcodon

Coding segment

Intron RNA

5 Cap

mRNA

Primary RNAPrimary RNAtranscripttranscript(pre-mRNA)(pre-mRNA)

5Exon

#2 & #3

#1

#2

#3

#4 #5

#6

To initiate transcription, eukaryotic RNA polymerase requires the

assistance of proteins called transcription factors (TFs)

– General TFs 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 TFs

proximal control elements-are located close to the promoter

distal control elements- groups of which are called enhancers, may be far away from a gene or even in an intron

activator- specific TF,

a protein that binds to an enhancer & stimulates transcription of a gene

repressor- specific TF, inhibit expression of a gene

#2

Activator proteins bindto distal control elementsgrouped as an enhancer in the DNA. This enhancer hasthree binding sites.

A DNA-bending protein brings the bound activators closer to the promoter. Other transcription factors, mediator proteins, and RNA polymerase are nearby.

The activators bind to certain general transcription factors and mediator proteins, helping them form an active transcription initiation complex on the promoter.

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

Combinatorial Control of Gene Activation

A particular combination of control elements can activatetranscription only when theappropriate activator proteinsare present

#2

Coordinately Controlled Genes

• 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

#2

Mechanisms of Post-Transcriptional Regulation

• Transcription alone does not account for gene expression

• More and more examples are being found of regulatory mechanisms that operate at various stages after transcription

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

#3-6

or

RNA splicing

mRNA

PrimaryRNA transcript

Troponin T gene

Exons

DNA

Alternative RNA Splicing

different mRNA molecules are produced

from the same primary transcript,

depending on which RNA segments are treated as exons/introns

#3

mRNA Degradation #4

• 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

Initiation of Translation

• The initiation of translation of selected mRNAs– can be blocked by regulatory proteins that bind to

specific 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

#5

Protein tobe degraded

Ubiquitinatedprotein

Proteasome

Protein entering aproteasome

Protein fragments(peptides)

Proteasomeand ubiquitinto be recycled

Ubiquitin

Protein Processing and Degradation• After translation, various types of protein processing,

including cleavage and the addition of chemical groups, are subject to control

proteasomes-

are giant protein complexes that bind protein molecules and degrade them

#1

#2

#3

#4

#6Multiple ubiquitin molecules are attached to a protein by enzymes in the cytosol.

The ubiquitin-tagged proteinis recognized by a proteasome,which unfolds the protein andsequesters it within a central cavity.

Enzymatic components of the proteasome cut the protein into small peptides, which can be further degraded by other enzymes in the cytosol.

#5

#6

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 forliver-specific genes

Enhancer forlens-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

REVIEW

Noncoding RNAs play multiple roles in controlling gene expression

• Only a small fraction of DNA codes for proteins, and a very small fraction of the non-protein-coding DNA consists of genes for RNA such as rRNA and tRNA

• A significant amount of the genome may be transcribed into noncoding RNAs (ncRNAs)

• Noncoding RNAs regulate gene expression at two points: • mRNA translation• chromatin configuration

(a) Primary miRNA transcript

HairpinmiRNA

miRNA

Hydrogenbond

Dicer

miRNA-proteincomplex

mRNA degraded Translation blocked

(b) Generation and function of miRNAs

5 3

• MicroRNAs (miRNAs)-

small single-stranded RNA molecules that can bind to mRNA• These can degrade mRNA

or block its translation

• The phenomenon of inhibition of gene expression by RNA molecules is called

RNA interference (RNAi)

• RNAi is caused by

small interfering RNAs (siRNAs)

• siRNAs and miRNAs are similar but form from different RNA precursors

• In some yeasts siRNAs play a role in heterochromatin formation and can block large regions of the chromosome

• RNA-based mechanisms may also block transcription of single genes

Chromatin modification

Transcription

RNA processing

mRNAdegradation

Translation

Protein processingand degradation

Chromatin modification

Translation

mRNA degradation

• miRNA or siRNA can target specificmRNAs for destruction.

• miRNA or siRNA can block the translationof specific mRNAs.

• Small or large noncoding RNAs canpromote the formation of heterochromatinin certain regions, blocking transcription.

REVIEW