Chapter 18~Regulaton of Gene Expression

2007-2008

Control of Prokaryotic (Bacterial) Genes

Bacterial metabolism• Bacteria need to respond quickly to changes in

their environment– if they have enough of a product,

need to stop production• why? waste of energy to produce more• how? stop production of enzymes for synthesis

– if they find new food/energy source, need to utilize it quickly• why? metabolism, growth, reproduction• how? start production of enzymes for digestion

STOP

GO

Remember Regulating Metabolism?• Feedback inhibition– product acts

as an allosteric inhibitor of 1st enzyme in tryptophan pathway

– but this is wasteful production of enzymes

= inhibition-

-Oh, I remember thisfrom our Metabolism Unit!

Different way to Regulate Metabolism• Gene regulation – instead of blocking

enzyme function, block transcription of genes for all enzymes in tryptophan pathway• saves energy by

not wasting it on unnecessary protein synthesis

= inhibition-

Now, that’s a good idea from a lowly bacterium!

--

Gene regulation in bacteria• Cells vary amount of specific enzymes by

regulating gene transcription– turn genes on or turn genes off• turn genes OFF example

if bacterium has enough tryptophan then it doesn’t need to make enzymes used to build tryptophan• turn genes ON example

if bacterium encounters new sugar (energy source), like lactose, then it needs to start making enzymes used to digest lactose

STOP

GO

Bacteria group genes together • Operon – genes grouped together with related functions

• example: all enzymes in a metabolic pathway– promoter = RNA polymerase binding site

• single promoter controls transcription of all genes in operon• transcribed as one unit & a single mRNA is made

– operator = DNA binding site of repressor protein

So how can these genes be turned off?• Repressor protein– binds to DNA at operator site – blocking RNA polymerase– blocks transcription

operatorpromoter

Operon model

DNATATA

RNApolymerase

repressor

repressor = repressor protein

Operon: operator, promoter & genes they controlserve as a model for gene regulation

gene1 gene2 gene3 gene4RNApolymerase

Repressor protein turns off gene by blocking RNA polymerase binding site.

1 2 3 4mRNA

enzyme1 enzyme2 enzyme3 enzyme4

mRNA

enzyme1 enzyme2 enzyme3 enzyme4operatorpromoter

Repressible operon: tryptophan

DNATATA

RNApolymerase

tryptophan

repressor repressor protein

repressortryptophan – repressor proteincomplex

Synthesis pathway modelWhen excess tryptophan is present, it binds to tryp repressor protein & triggers repressor to bind to DNA– blocks (represses) transcription

gene1 gene2 gene3 gene4

conformational change in repressor protein!

1 2 3 4

repressortrpRNApolymerase

trp

trp

trp trp

trp trp

trptrp

trptrp

trp

Tryptophan operonWhat happens when tryptophan is present?Don’t need to make tryptophan-building enzymes

Tryptophan is allosteric regulator of repressor protein

mRNA

enzyme1 enzyme2 enzyme3 enzyme4operatorpromoter

Inducible operon: lactose

DNATATARNApolymerase

repressor repressor protein

repressorlactose – repressor proteincomplex

lactose

lac repressor gene1 gene2 gene3 gene4

Digestive pathway model When lactose is present, binds to lac repressor protein & triggers repressor to release DNA– induces transcription

RNApolymerase

1 2 3 4

lac lac

laclac

laclac

lac

conformational change in repressor protein!

lac

lac

Lactose operonWhat happens when lactose is present?Need to make lactose-digesting enzymes

Lactose is allosteric regulator of repressor protein

Jacob & Monod: lac Operon• Francois Jacob & Jacques Monod– first to describe operon system– coined the phrase “operon”

1961 | 1965

Francois JacobJacques Monod

Operon summary• Repressible operon – usually functions in anabolic pathways

• synthesizing end products

– when end product is present in excess,cell allocates resources to other uses

• Inducible operon – usually functions in catabolic pathways,

• digesting nutrients to simpler molecules

– produce enzymes only when nutrient is available• cell avoids making proteins that have nothing to do, cell

allocates resources to other uses

Positive gene control

• occurs when an activator molecule interacts directly with the genome to switch transcription on.

• Even if the lac operon is turned on by the presence of allolactose, the degree of transcription depends on the concentrations of other substrates.

• The cellular metabolism is biased toward the utilization of glucose.

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

Positive Gene Regulation– If glucose levels are

low (along with overall energy levels), then cyclic AMP (cAMP) binds to cAMP receptor protein (CRP) which activates transcription.

• If glucose levels are sufficient and cAMP levels are low (lots of ATP), then the CRP protein has an inactive shape and cannot bind upstream of the lac promotor.

2007-2008

Control of Eukaryotic Genes

The BIG Questions…• How are genes turned on & off

in eukaryotes?• How do cells with the same genes

differentiate to perform completely different, specialized functions?

Evolution of gene regulation• Prokaryotes– single-celled– evolved to grow & divide rapidly– must respond quickly to changes in external

environment• exploit transient resources

• Gene regulation– turn genes on & off rapidly• flexibility & reversibility

– adjust levels of enzymes for synthesis & digestion

Evolution of gene regulation• Eukaryotes– multicellular– evolved to maintain constant internal

conditions while facing changing external conditions• homeostasis

– regulate body as a whole• growth & development

– long term processes• specialization

– turn on & off large number of genes• must coordinate the body as a whole rather than

serve the needs of individual cells

Points of control• The control of gene expression

can occur at any step in the pathway from gene to functional protein1. packing/unpacking DNA

2. transcription

3. mRNA processing

4. mRNA transport

5. translation

6. protein processing

7. protein degradation

1. DNA packing as gene control• Degree of packing of DNA regulates transcription– tightly wrapped around histones

• no transcription• genes turned off heterochromatin

darker DNA (H) = tightly packed euchromatin

lighter DNA (E) = loosely packed

H E

DNA methylation• Methylation of DNA blocks transcription factors – no transcription

genes turned off– attachment of methyl groups (–CH3) to cytosine

• C = cytosine– nearly permanent inactivation of genes

• ex. inactivated mammalian X chromosome = Barr body

Histone acetylation Acetylation of histones unwinds DNA

loosely wrapped around histones enables transcription genes turned on

attachment of acetyl groups (–COCH3) to histones

conformational change in histone proteins transcription factors have easier access to genes

Epigenetic Inheritance

• 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

2. Transcription initiation

• Control regions on DNA– promoter• nearby control sequence on DNA• binding of RNA polymerase & transcription factors• “base” rate of transcription

– enhancer• distant control

sequences on DNA• binding of activator

proteins• “enhanced” rate (high level)

of transcription

Model for Enhancer action

• Enhancer DNA sequences – distant control sequences

• Activator proteins – bind to enhancer sequence &

stimulates transcription

• Silencer proteins – bind to enhancer sequence & block

gene transcription

Turning on Gene movie

Transcription complex

Enhancer

ActivatorActivator

Activator

Coactivator

RNA polymerase II

A

B F E

HTFIID

Core promoterand initiation complex

Activator Proteins• regulatory proteins bind to DNA at distant

enhancer sites• increase the rate of transcription

Coding region

T A T A

Enhancer Sitesregulatory sites on DNA distant from gene

Initiation Complex at Promoter Site binding site of RNA polymerase

Fig. 18-9-3

Enhancer TATAbox

PromoterActivators

DNAGene

Distal controlelement

Group ofmediator proteins

DNA-bendingprotein

Generaltranscriptionfactors

RNApolymerase II

RNApolymerase II

Transcriptioninitiation complex RNA synthesis

3. Post-transcriptional control• Alternative RNA splicing– variable processing of exons creates a family of

proteins

4. Regulation of mRNA degradation• Life span of mRNA determines amount of

protein synthesis– mRNA can last from hours to weeks

RNA processing movie

5. Control of translation• Block initiation of translation stage – regulatory proteins attach to 5' end of mRNA • prevent attachment of ribosomal subunits & initiator

tRNA• block translation of mRNA to protein

Control of translation movie

6-7. Protein processing & degradation

• Protein processing– folding, cleaving, adding sugar groups,

targeting for transport• Protein degradation– ubiquitin tagging– proteasome degradation

Protein processing movie

Ubiquitin• “Death tag”– mark unwanted proteins with a label – 76 amino acid polypeptide, ubiquitin– labeled proteins are broken down rapidly in

"waste disposers"• proteasomes

1980s | 2004

Aaron CiechanoverIsrael

Avram HershkoIsrael

Irwin RoseUC Riverside

Proteasome • Protein-degrading “machine”– cell’s waste disposer– breaks down any proteins

into 7-9 amino acid fragments• cellular recycling

play Nobel animation

Concept 18.3: 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

RNA interference• Small interfering RNAs (siRNA)– short segments of RNA (21-28 bases)• bind to mRNA• create sections of double-stranded mRNA• “death” tag for mRNA– triggers degradation of mRNA

– cause gene “silencing”• post-transcriptional control• turns off gene = no protein produced

NEW!

siRNA

Action of siRNA

siRNA

double-stranded miRNA + siRNA

mRNA degradedfunctionally turns gene off

Hot…Hotnew topicin biology

mRNA for translation

breakdownenzyme(RISC)

dicerenzyme

initiation of transcription

1

mRNA splicing

2

mRNA protection3

initiation of translation

6

mRNAprocessing

5

1 & 2. transcription - DNA packing - transcription factors

3 & 4. post-transcription - mRNA processing

- splicing- 5’ cap & poly-A tail- breakdown by siRNA

5. translation - block start of translation

6 & 7. post-translation - protein processing - protein degradation

7 protein processing & degradation

4

4

Gene Regulation

Molecular Biology of Cancer• Oncogene

•cancer-causing genes• Proto-oncogene

•normal cellular genes• How?

1-movement of DNA; chromosome fragments that have rejoined incorrectly 2-amplification; increases the number of copies of proto-oncogenes

• 3-proto-oncogene point mutation; protein product more active or more resistant to degradation

• Tumor-suppressor genes •changes in genes that prevent uncontrolled cell growth (cancer growth stimulated by the absence of suppression)

Cancers result from a series of genetic changes in a cell lineage

– The incidence of cancer increases with age because multiple somatic mutations are required to produce a cancerous cell

– As in many cancers, the development of colon cancer is gradual