Lecture 18 6

8/11/2019 Lecture 18 6

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Today

More operonsPhage Lambda

Arabinose Operon ( ara )

• encodes genes for theutilization of another sugar,arabinose

• as was the case with lactose,cell prefers to use glucose if itis available

• the ara operon responds toboth arabinose and glucose

Structure of ara operon

• 3 structural genes: araB , araA and araD• CAP-bindng site• inducer binding site ( araI )• operator ( araO )• araC (not part of ara operon) encodes the

AraC protein, which exerts both positiveand negative control of the ara operon

ara operon

Positive regulation of ara operon

If arabinose is present :• arabinose binds to AraC protein• AraC-arabinose binds to the araI site (the inducer site)

…and glucose is absent:• CAP-cAMP binds to the CAP site• RNA polymerase binds ara BAD promoter • Genes for arabinose enzymes B, A, and D are

transcribed, translated, arabinose is metabolized

ara operoninduced

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8/11/2019 Lecture 18 6


Back to Chapter 16

Repressible Operon

• anabolism – synthesis• anabolic operons: turned on when amount

of a synthesis product is low, turned off(repressed) when amount is high

• two mechanisms of control – negative regulation by repressor proteins – attenuation : regulation by changes in

secondary structure of mRNA

trp operon - Repression

• five structural genes encode enzymes forsynthesis of amino acid tryptophan

• promoter ( trpP ), operator ( trpO ) and regulatorygene ( trpR )

• product of trpR is repressor, TrpR• TrpR binds tryptophan (corepressor)• tryptophan abundant: TrpR-tryptophan binds to

operator and represses transcription• tryptophan scarce: tryptophan leaves TrpR,

TrpR leaves operator, operon is derepressed

trp operon - Attenuation

• attenuator region between operator andfirst structural gene ( trpE )

• mRNA transcribed from attenuator (leadertranscript) has four subregions that form 3different stem-loop structures

• if stem-loops with regions 1-2 and 3-4form, stem-loops with regions 2-3 can’tform

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trp operon leader peptide

• leader transcript also contains a short

peptide-encoding gene with two adjacenttryptophan codons

• region 1 is located within the leadertranscript

trp Operon Attenuation of trp

Abundant tryp tophan :• ribosome translates leader

peptide quickly, position ofribosome causes region 3-4stem-loop to form

• Region 3-4 stem-loop structureacts as a terminator , stopstranscription

Attenuation of trp

Low tryptophan:• ribosome pauses on tryptophan

codons in leader sequence andprevents region 1 from pairing with

region 2• region 2-3 stem-loop formsinstead, which prevents region 3-4stem loop (the terminator) fromforming

• no termination, transcriptioncontinues

Attenuation of trp

General starvation fo ramino acids:

• ribosome stalls at beginning ofleader, 1-2 and 3-4 stem-loopstructures form, 3-4 structure isterminator

• stops transcription

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TRAP Regulation of trp Operon inBacillis subtilis

• Attenuation, but details differ from E. coli

• Binding of TRAP protein (trp RNA binding attenuationprotein) determines which stem-loop structures form• TRAP has 11 identical subunits, which bind to 11

tryptophan molecules and 11 triplets in mRNA• tryptophan levels are high :

TRAP binding prevents regions A and B in mRNA from base-pairing, instead C and D base-pair and form a -independentterminator

• tryptophan levels are low:TRAP doesn’t bind to RNA and an antiterminator stem-loopforms instead of terminator

TRAP binding tryptophan and RNA

TRAP protein and attenuationPhage Lambda

Lysis vs . Lysogeny

Lysis vs. Lysogeny

• Lysis : Infection by phage produces manyprogeny and breaks open (lyses) the hostbacterium

• Lysogeny : After infection, the phage DNAintegrates into the host genome and residesthere passively – No progeny – No lysis of the host

• Bacteriophage lambda can do either.

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Lysogeny

• The phage genome integrated into the host

bacterial genome is a prophage .• Bacterium carrying the prophage is a lysogen .• Lysogens are immune to further infection by

similar phage because the phage functions arerepressed in trans .

• Induction of the lysogen leads to excision ofthe prophage, replication of the phage DNA,and lysis of the host bacterium.

Organization of DNA

• Genes are organized into operons

• Left operon – genes that prepare for lysogeny

(recombination and integration)

• Right operon – genes that prepare fo rlysis

• Late operon – genes for phage head and tail proteins, lysis

after infection

• DNA is linear in phage head• DNA circularizes shortly after infection, by

base-pairing at single-stranded cos sites• Host RNA polymerase begins to transcribe

immediate early phage genes

After infection phage can be eitherlytic or lysogenic.

• Lysis : Infection by phage produces manyprogeny and breaks open (lyses) the hostbacterium

• Lysogeny : After infection, the phage DNAintegrates into the host genome and residesthere passively – No progeny – No lysis of the host

Lysis or lysogeny is determined by a contestbetween two genes that encode repressors.

cI gene

encodes the repressor that blockstranscription of all other genescr o gene

encodes a repressor that blockstranscription of cI gene and allows othergenes to be transcribed

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Lyis or Lysogeny?

Lytic and lysogenic pathways start out the

same, then diverge after expression of thedelayed early genes.

We will trace the lytic pathway first.

Immediate early genes are expressed first

N gene from left promoter (P L)(transcribed to left)

cr o gene from right promoter (P R)(transcribed to right)

Further transcription is blocked bydependent terminators .

c I gene is in between promoters,not transcribed at first

N gene product is an antiterminator thatallows transcription of delayed early genes

• Delayed early genes transcribed from P L

– genes for integration into host (lysogeny)

• … from P R

– genes for DNA replication (lysis)

N protein (green) overcomesterminators (red)

Delayed Early GenesDelayed early genes transcribed from P L and P RTwo delayed early genes allow theexpression of late genes for lysis .

• cr o gene product is an "antirepressor“ – represses the repressor ( cI gene product)

• Q gene product is an antiterminator – allows transcription of late genes

• If phage goes into lytic mode, late genesare transcribed from P R'

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repression by cro product

antitermination by Q product

Transcription from P R' of lategenes for lysis

The lysogenic pathway begins with theexpression of two delayed early genes.

Establishment of lysogeny begins withexpression of cII and cIII

cIII encodes a protein thatinhibits a protease thatcan degrade the cIIprotein

cII protein (if not degraded)increases transcription atP RE and allowstranscription of cI gene

The cI gene product is the repressor, which isneeded to establish and maintain lysogeny.

The repressor – represses early gene promoters, thereby

repressing cro and preventing lysis – increases transcription from its own promoter,

thereby maintaining lysogeny

Delayed early gene products from P L andthe repressor establish lysogeny.

• Delayed early gene products allowintegration of phage into host genome

• Expression of cI (repressor) preventstranscription of all genes except itself.

Expression of repressor during lysogenymaintains lysogeny .

• Continued expression of cI (repressor)prevents transcription of other phagegenes.

• If another phage enters the cell, therepressor blocks its transcription

• Every time the host divides, the prophageis replicated with it.

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When to establish lysogeny?

• If host cell doesn’t have enough resources

to become a “phage factory”, wait forbetter times.

• If cell growth has been limited, lysogeny isfavored.

• Activity of host cell’s proteases is used forthis decision.

Phage senses amount of cell growth byhow much cII protein is degraded.

If E. coli cell growth is limited , proteases arelimited- cII is less likely to get degraded- Enhances cI transcription- Lysogeny is favored

If E. coli is experiencing active bacterial cellgrowth, more proteases- cII is more likely to be degraded, so that itcan’t help cI get transcribed- Lysis is favored

When to leave lysogeny and enter lyticmode?

If host cell is damaged, better to enter lyticmode before the cell dies.

UV damage to host leads to lysogeny

• UV damage induces DNA repair – SOS system – recA gene involved in recombination, also

works as a protease – Protease RecA* degrades protein

(repressors)• Action of RecA* allows repressed operons to

be activated• cI cleaved, cro can bind operator• Result of UV damage: lysis

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Lecture 18 6