Control of Gene

Expression

AP Chap 18

Overview: Conducting the Genetic Orchestra

• Prokaryotes and eukaryotes alter gene expression in response to their changing environment.

Bacteria often respond to environmental change by regulating transcription

• Natural selection has favored bacteria that produce only the products needed by that cell.

• A cell can regulate the production of enzymes by feedback inhibition or by gene regulation.

• Gene expression in bacteria is controlled by the operon model.

Fig. 18-2

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

Precursor

Feedbackinhibition

OPERONS

• An operon is the entire stretch of DNA that includes the operator, the promoter, and the genes that they control

regulator promoter operator genes

regulator promoter operator genes

Makes arepressorwhich bindsto operatorand stops/startstranscription

RNA polymerasebinds here

Induction System

• System initially off• The presence of an inducer (usually a

substrate that needs to be broken down) turns it on.

• The inducer binds to the repressor and makes it inactive so transcription can occur.

• The inducer acts as an allosteric effector and changes the shape of the repressor.

• Ex- Lac (lactose) operon

Fig. 18-4a

(a) Lactose absent, repressor active, operon off

DNA

ProteinActiverepressor

RNApolymerase

Regulatorygene

Promoter

Operator

mRNA5

3

NoRNAmade

lacI lacZ

Fig. 18-4b

(b) Lactose present, repressor inactive, operon on

mRNA

Protein

DNA

mRNA 5

Inactiverepressor

Allolactose(inducer)

5

3RNApolymerase

Permease Transacetylase

lac operon

-Galactosidase

lacYlacZ lacAlacI

Lactose present, repressor inactive, operon ON

Repressible System

• System initially ON, transcription ongoing and making a product

• Operator can be turned off by a repressor which is made active by being activated by a corepressor molecule (usually the end product)

• Ex – tryptophan operon – trytophan acts as a corepressor inhibitng further synthesis of enzymes involved in the process

Fig. 18-3a

Polypeptide subunits that make upenzymes for tryptophan synthesis

(a) Tryptophan absent, repressor inactive, operon on

DNA

mRNA 5

Protein Inactiverepressor

RNApolymerase

Regulatorygene

Promoter Promoter

trp operon

Genes of operon

OperatorStop codonStart codon

mRNA

trpA

5

3

trpR trpE trpD trpC trpB

ABCD

E

Tryptophan absent, repressor inactive, operon ON

Fig. 18-3b-1

(b) Tryptophan present, repressor active, operon off

Tryptophan(corepressor)

No RNA made

Activerepressor

mRNA

Protein

DNA

Tryptophan present, repressor active, operon OFF

INDUCIBLE REPRESSABLE

OFF ON turned on by turned off by inducer corepressor used in catabolic used in anabolic pathways pathways

Both use allosteric effectors and are NEGATIVE CONTROL.

Positive Gene Regulation

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

• When glucose (a preferred food source of E. coli) is scarce, CAP is activated by binding with cyclic AMP

• Activated CAP attaches to the promoter of the lac operon and increases the affinity of RNA polymerase, thus accelerating transcription

Fig. 18-5

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

cAMP

DNA

Inactive lacrepressor

Allolactose

InactiveCAP

lacI

CAP-binding site

Promoter

ActiveCAP

Operator

lacZ

RNApolymerasebinds andtranscribes

Inactive lacrepressor

lacZ

OperatorPromoter

DNA

CAP-binding site

lacI

RNApolymerase lesslikely to bind

InactiveCAP

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

• When glucose levels increase, CAP detaches from the lac operon, and transcription returns to a normal rate

• CAP helps regulate other operons that encode enzymes used in catabolic pathways

Control of Gene Expression in Eukaryotes

• In response to environmental signals

• Essential for development and cell specialization in multicellular organisms

• RNA is important in gene expression.

• All cells contain the same DNA so controlling gene expression is essential.

• Human cells only 20% genes expressed; only 1.5% code for proteins

• Commonly occurs at level of transcription; hence, gene expression = transcription of DNA

Eukaryotic gene expression can be regulated at any stage

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Fig. 18-6

DNA

Signal

Gene

NUCLEUS

Chromatin modification

Chromatin

Gene availablefor transcription

Exon

Intron

Tail

RNA

Cap

RNA processing

Primary transcript

mRNA in nucleus

Transport to cytoplasm

mRNA in cytoplasm

Translation

CYTOPLASM

Degradationof mRNA

Protein processing

Polypeptide

Active protein

Cellular function

Transport to cellulardestination

Degradationof protein

Transcription

Fig. 18-6a

DNA

Signal

Gene

NUCLEUS

Chromatin modification

Chromatin

Gene availablefor transcription

Exon

Intron

Tail

RNA

Cap

RNA processing

Primary transcript

mRNA in nucleus

Transport to cytoplasm

CYTOPLASM

Transcription

Fig. 18-6b

mRNA in cytoplasm

Translation

CYTOPLASM

Degradationof mRNA

Protein processing

Polypeptide

Active protein

Cellular function

Transport to cellulardestination

Degradationof protein

1) State of chromatin (nucleosomes)

• Heterochromatin – genes not expressed due to tightly wound DNA

Chemical modification

- Histone acetylation – promotes transcription by inhibiting binding between nucleosomes so keeps DNA spread out; also may recruit transcription factors

- Methylation of DNA – inhibits transcription (indicated in inactive X)

Epigenetic inheritance – not involved DNA sequence but inherited defects in chromatin modification enzymes

There may be more to inheritance than genes alone. New clues reveal that a second epigenetic chemical code sits on top of our regular DNA and controls how our genes are expresse.

2) Transcription Level

• Remember, transcription factors bind to DNA promoters, then RNA polymerase binds to promoter region to form the Transcription Initiation Complex

Transcription Factors:General

Bind to RNA polymerase and each other to initiate transcription of all protein-coding genes

Low rate of transcription

Specific Transcription Factors

• (a) enhancers: proximal (close to promoters) and distal; specific for a gene

• (b) some are repressors and can block anywhere in the scheme and can also recruit proteins to affect chromatin structure

• (c) enhancers contain control elements (up to 10 different ones); the combination is specific for transcription factors. Also explains how genes in a related pathway are correlated (like flags on mailboxes to know which mail to pick up). The combination signals which genes are expressed.

Fig. 18-9-1

Enhancer TATAbox

PromoterActivators

DNAGene

Distal controlelement

Fig. 18-9-2

Enhancer TATAbox

PromoterActivators

DNAGene

Distal controlelement

Group ofmediator proteins

DNA-bendingprotein

Generaltranscriptionfactors

Fig. 18-9-3

Enhancer TATAbox

PromoterActivators

DNAGene

Distal controlelement

Group ofmediator proteins

DNA-bendingprotein

Generaltranscriptionfactors

RNApolymerase II

RNApolymerase II

Transcriptioninitiation complex RNA synthesis

Fig. 18-UN7

Fig. 18-10

Controlelements

Enhancer

Availableactivators

Albumin gene

(b) Lens cell

Crystallin geneexpressed

Availableactivators

LENS CELLNUCLEUS

LIVER CELLNUCLEUS

Crystallin gene

Promoter

(a) Liver cell

Crystallin genenot expressed

Albumin geneexpressed

Albumin genenot expressed

3. Post-transcriptional Control

• Alternative RNA splicing (mRNA can last a long time and be subject to various intron splicing)

•The mRNA life span is determined in part by sequences in the leader and trailer regions

Fig. 18-11

or

RNA splicing

mRNA

PrimaryRNAtranscript

Troponin T gene

Exons

DNA

• Alteration of polypeptide - can be cut, groups added, or transported to target locations

• Selective degradation of proteins – the protein ubiquitin are added to proteins for degradation. Proteasomes recognize them and destroy them.

Fig. 18-12

Proteasomeand ubiquitinto be recycledProteasome

Proteinfragments(peptides)Protein entering a

proteasome

Ubiquitinatedprotein

Protein tobe degraded

Ubiquitin

Noncoding RNAs play multiple roles in controlling gene expression

• Only a small fraction of DNA codes for proteins, rRNA, and tRNA

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

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

Effects on mRNAs by MicroRNA’s

• MicroRNAs (miRNAs) are small single-stranded RNA molecules that can bind to mRNA

• These can degrade mRNA or block its translation

Hairpins?

• Long RNA precursors fold on themselves and look like hairpins. The hairpins are cut off and an enzyme called Dicer trims the ends. One strand becomes a microRNA (miRNA).

• These bind with proteins and block translation or degrades the mRNA.

Fig. 18-13

miRNA-proteincomplex(a) Primary miRNA transcript

Translation blocked

Hydrogenbond

(b) Generation and function of miRNAs

Hairpin miRNA

miRNA

Dicer

3

mRNA degraded

5

• An estimated 1/3 of human genes are regulated by miRNAs

Small Interfering RNA’s

• The phenomenon of inhibition of gene expression by RNA molecules is called RNA interference (RNAi)

• Cells cut up the RNA into small interfering RNAs (siRNAs) that can inhibit expression like miRNA’s

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

CANCER AND GENE EXPRESSIONCancer results from genetic changes

that affect cell cycle control

• Cancer can be caused by mutations to genes that regulate cell growth and division

- mutagens are chemicals, X-rays, tumor viruses in animals

Fig. 18-21c

(c) Effects of mutations

EFFECTS OF MUTATIONS

Cell cycle notinhibited

Protein absent

Increased celldivision

Proteinoverexpressed

Cell cycleoverstimulated

Oncogenes and Proto-Oncogenes

• Oncogenes are cancer-causing genes• Proto-oncogenes are the

corresponding normal cellular genes that are responsible for normal cell growth and division

Conversion of a proto-oncogene to an oncogene can lead to abnormal

stimulation of the cell cycle

• Amplification of normal growth-stimulating gene

• Translocation of growth gene under control of a more active promoter

• Point mutation in control element or gene itself to make a hyperactive or degradation resistent growth protein.

Fig. 18-20

Normal growth-stimulatingprotein in excess

Newpromoter

DNA

Proto-oncogene

Gene amplification:Translocation ortransposition:

Normal growth-stimulatingprotein in excess

Normal growth-stimulatingprotein in excess

Hyperactive ordegradation-resistant protein

Point mutation:

Oncogene Oncogene

within a control element within the gene

Tumor-Suppressor Genes• help prevent uncontrolled cell growth

• Tumor-suppressor proteins

–Repair damaged DNA

–Control cell adhesion

–Inhibit the cell cycle

How do cancer genes work?

• 30% cancers – ras proto-oncogene gene is mutated

Ras gene codes for a protein that stimlates the production of a cell cycle stimulating protein; mutations cause a hyperactive protein

• 50% cancers – p53 gene mutated; codes for a transcription factor for growth-inhibiting proteins. These proteins bind to a p21 gene whose product binds to CDK’s and halt cell cycle. It can also activate DNA repair genes or “suicide genes” if DNA can’t be repaired.

Fig. 18-21b

MUTATIONProtein kinases

DNA

DNA damagein genome

Defective ormissingtranscriptionfactor, suchas p53, cannotactivatetranscription

Protein thatinhibitsthe cell cycle

Activeformof p53

UVlight

(b) Cell cycle–inhibiting pathway

2

3

1

p53 gene and DNA repair

P53 and suicide genes

Multistep Model of Cancer Development

• More than one somatic mutation is needed

• Both alleles must be defective

• In some, genes for telomerase becomes activated and cells divided continually

Breat cancer gene

Inherited Predisposition and Other Factors Contributing to Cancer

• Individuals can inherit oncogenes or mutant alleles of tumor-suppressor genes

• Inherited mutations in the tumor-suppressor gene adenomatous polyposis coli are common in individuals with colorectal cancer

• Mutations in the BRCA1 or BRCA2 gene are found in at least half of inherited breast cancers

• Even if you have cancer genes, it does not mean you will have cancer.

• Genes can be modified by siRNA’s, epigenesis, and the environment