Chapter 21

Operons: Fine Control of Bacterial

Transcription

The E. coli genome contains over 3000 genes. Some of these are active all the

time because their products are in constant demand. But some of them are

turned off most of the time because their products are rarely needed. For

example, the enzymes required for the metabolism of the sugar arabinose would

be useful only when arabinose is present and when the organism’s favorite

energy source, glucose, is absent. Such conditions are not common, so the

genes encoding these enzymes are usually turned off. Why doesn’t the cell just

leave all its genes on all the time, so the right enzymes are always there to take

care of any eventuality? The reason is that gene expression is an expensive

process. It takes a lot of energy to produce RNA and protein. In fact, if all of an E.

coli cell’s genes were turned on all the time, production of RNAs and proteins

would drain the cell of so much energy that it could not compete with more effi

cient organisms. Thus, control of gene expression is essential to life.

Gene regulation is the device to insure that proteins are synthesized in exactly

the amounts they are needed and only when they are needed.

Transcriptional regulation:

gene expression is controlled by regulatory proteins

Negative regulation:

- A repressor protein inhibits transcription of a specific gene.

- In this case, inducer (antagonist of the repressor) is needed to allow transcription.

Positive regulation:

- Activator works to increase the frequency of transcription of an gene (operon)

Transcriptional regulation:

gene expression is controlled by regulatory proteins

Operons

Operons and the resulting transcriptional regulation of gene expression permit

prokaryotes to rapidly adapt to changes in the environment: new carbon sources,

lack of an amino acid, etc. The genes involved in the catabolism of the carbon

source or in the biosynthesis (anabolism) of the amino acid can be rapidly turned

on or turned off.

Negative control of expression of the Operon is the dominant case.

An Operon is a set of genes that are expressed in unison, ie they are

Coordinately expressed, together with the DNA control elements used for their

expression.

Usually, an Operon is a set of genes which are adjacent to each other on the

chromosome and are Coordinately Expressed at the transcription level via a

single mRNA molecule (polycistronic mRNA): Coordinate regulation.

A negatively-acting protein called the Repressor regulates expression of the

Operon by binding to an Operator DNA site near the promoter for transcription.

Expression of the gene encoding the Repressor is usually NOT itself controlled by

a Repressor. It then is expressed at all times, ie is constitutively expressed.

This Operator binding site for the Repressor is the major type of DNA control

element.

The genes that are Coordinately expressed are called Structural Genes and

encode proteins that function enzymatically in a common process such as a

biosynthetic or catabolic pathway.

The genes that encode regulatory proteins such as Repressors are called

Regulator Genes and are NOT usually part of the set of coordinately expressed

operon genes.

A small molecule (metabolite) usually interacts with the Repressor. This small

molecule is either an Inducer or a Co-Repressor:

An Inducer inactivates the Repressor in the Inducer-Repressor complex.

A Co-Repressor activates the Repressor in the Inducer-Repressor complex.

Thus binding of this small molecule to one site of Repressor alters its ability to bind

Operator DNA at a second site on Repressor ... example of Allosterism

NOTE: Products of Regulatory Genes can then diffuse or move to their targets and

hence are Trans-acting Factors.

Operators and DNA control elements are the targets and affect only the DNA to

which they are attached; they are Cis-acting elements. Any protein binding site on

DNA, e.g. transcription promoters and terminators, are cis-acting elements.

Operons

Two general Operon Classes: Catabolic and Anabolic Pathways

Catabolic Operons:

The small molecule interacting with the Repressor is an Inducer, and is usually the sugar or

metabolic which is broken down.

Note the rationale: the operon should be expressed only when Inducer is present, to express

the genes needed to break down (catabolize) the Inducer, e.g., sugar

General Example:

Structural genes A, B, C with Operator O near Promoter P.

Regulator gene R constitutively expressing Repressor R inactivated by Inducer I, as shown

in the following Figure:

Two general Operon Classes: Catabolic and Anabolic Pathways

Anabolic (biosynthetic) Operons:

The small molecule interacting with the Repressor is a Co-Repressor, and is usually

the end-product of the biosynthetic pathway, e.g., an amino acid.

Note the rationale: the operon should be expressed only when Co-Repressor is

absent, to express the genes needed to synthesize the Co-Repressor, e.g., amino

acid

General Example:

Structural genes A, B, C with Operator O near Promoter P.

Regulator gene R constitutively expressing Repressor R activated by Co-Repressor

Co, as shown in the following Figure:

7.1 The lac Operon • The lac operon was the first operon discovered

• It contains 3 genes coding for E. coli proteins that permit the bacteria to use the sugar lactose

– Galactoside permease (lacY) which transports lactose into the cells

b-galactosidase (lacZ) cuts the lactose into galactose and glucose

– Galactoside transacetylase (lacA) whose function is unclear

• All 3 genes are transcribed together producing 1 mRNA, a polycistronic

message that starts from a single promoter

– Each cistron, or gene, has its own ribosome binding site

– Each cistron can be translated by separate ribosomes that bind

independently of each other

Control of the lac Operon

• The lac operon is tightly controlled, using 2 types of control – Negative control, like the brake of a car, must remove the repressor

from the operator - the “brake” is a protein called the lac repressor

– Positive control, like the accelerator pedal of a car, an activator, additional positive factor responds to low glucose by stimulating transcription of the lac operon

• Negative control indicates that the operon is turned on unless

something intervenes and stops it

• The off-regulation is done by the lac repressor

– Product of the lacI gene

– Tetramer of 4 identical polypeptides

– Binds the operator just right of promoter

Negative Control of the lac Operon

• When the repressor binds to the operator, the operon is repressed

– Operator and promoter sequence are contiguous

– Repressor bound to operator prevents RNA polymerase from binding to

the promoter and transcribing the operon

• As long as no lactose is available, the lac operon is repressed

Inducer of the lac Operon

• The repressor is an allosteric protein

– Binding of one molecule to the protein changes shape of a remote

site on that protein

– Altering its interaction with a second molecule

• The inducer binds the repressor

– Causing the repressor to change conformation that favors release

from the operator

• The inducer is allolactose, an alternative form of lactose

Discovery of the Operon

During the 1940s and 1950s, Jacob and Monod studied the metabolism of lactose by E. coli

•Three enzyme activities / three genes were induced together by galactosides

• Constitutive mutants need no induction, genes are active all the time

• Created merodiploids or partial diploid bacteria carrying both wild-type (inducible) and constitutive alleles

(Partial diploid=meroploid)

Lac+ phenotype

F'lacY-lacZ+/ lacY+lacZ-

F'lacY+lacZ-/ lacY-lacZ+

Lac- phenotype

F'lacY-lacZ+/ lacY-lacZ+

F'lacY+lacZ-/ lacY+lacZ-

Discovery of the Operon

• Using merodiploids or partial diploid bacteria carrying both

wild-type and constitutive alleles distinctions could be made by

determining whether the mutation was dominant or recessive

• Because the repressor gene produces a repressor protein

that can diffuse throughout the nucleus, it can bind to both

operators in a meriploid and is called a trans-acting gene

because it can act on loci on both DNA molecules

• Because an operator controls only the operon on the same

DNA molecule it is called a cis-acting gene

Effects of Regulatory Mutations:

(a) Wild-type and Mutated Repressor

(b) Wild-type and Mutated Operator with Defective Binding

Effects of Regulatory Mutations: (c& d) Wild-type and Mutated Operon binding Irreversibly

Lac operon

Constitutive

mutation

trans-acting factor, cis-acting element

Repressor-Operator Interactions

• Using a filter-binding assay, the lac repressor binds to the

lac operator

• A genetically defined constitutive lac operator has lower

than normal affinity for the lac repressor

• Sites defined by two methods as the operator are in fact the

same

Assaying the binding between lac

operator and lac repressor. Cohn and

colleagues labeled lacO-containing DNA

with 32P and added increasing amounts of

lac repressor. They assayed binding

between repressor and operator by

measuring the radioactivity attached to

nitrocellulose. Only labeled DNA bound

to repressor would attach to nitrocellulose.

Red: repressor bound in the absence of

the inducer IPTG. Blue: repressor bound

in the presence of 1 mM IPTG, which

prevents repressor–operator binding.

The Mechanism of Repression

• The repressor does not block access by

RNA polymerase to the lac promoter

• Polymerase and repressor can bind

together to the lac promoter

• Polymerase-promoter complex is in

equilibrium with free polymerase and

promoter

lac Repressor and Dissociation of RNA

Polymerase from lac Promoter

• Without competitor,

dissociated polymerase

returns to promoter

• Heparin and repressor

prevent reassociation of

polymerase and promoter

• Repressor prevents

reassociation by binding to

the operator adjacent to the

promoter

• This blocks access to the

promoter by RNA

polymerase

Mechanism Summary

• Two competing hypotheses of mechanism for

repression of the lac operon

– RNA polymerase can bind to lac promoter in

presence of repressor

• Repressor will inhibit transition from abortive transcription to

processive transcription

– The repressor, by binding to the operator, blocks

access by the polymerase to adjacent promoter.

– The latest evidence supports the latter hypothesis.

lac Operators

• There are three lac operators

– The major lac operator lies adjacent to promoter

– Two auxiliary lac operators - one upstream and the other

downstream

• All three operators are required for optimum repression

• The major operator produces only a modest amount of

repression

Catabolite Repression of the lac Operon

• Function of Catabolite Repression: Facilitate use of Glucose over

Lactose (or other sugars) when both Glucose and Lactose are available

to the bacteria

In media, glucose+lactose (or galactose) >> glucose consumption

Lactose, lactose (by lac operon) +galactose (by gal operon)

• When glucose is present, the lac operon is in a relatively inactive state

• Selection in favor of glucose attributed to role of a breakdown product,

catabolite

• Process known as catabolite repression uses a breakdown product to

repress the operon

Catabolite Repression of the lac Operon • Function of Catabolite Repression: Facilitate use of Glucose over Lactose (or

other sugars) when both Glucose and Lactose are available to the bacteria

In media, glucose+lactose (or galactose) >> glucose consumption

Lactose, lactose (by lac operon) +galactose (by gal operon)

• When glucose is present, the lac operon is in a relatively inactive state

cAMP is synthesized by the enzyme adenylate cylase, and its concentration is

related to glucose concentration. So,

Mechanism:

• When Glucose is present, cAMP (3',5'-cyclic-AMP) levels are low, little CAP-cAMP

complex is formed, and low expression of Lac operon even when Lactose is

present. (CAP: catabolite Activator protein= CRP (cAMP receptor protein)

• When Glucose is absent, cAMP levels are high, much CAP-cAMP complex is

formed, and Lactose induces Lac operon to high level of expression

Glucose >> cAMP and Glucose >> cAMP

Catabolite Activator Protein

• cAMP added to E. coli can overcome catabolite repression of the lac operon

• The addition of cAMP leads to the activation of the lac gene even in the presence of glucose

• Positive controller of lac operon has 2 parts: – cAMP

– A protein factor is known as:

• Catabolite activator protein or CAP

• Cyclic-AMP receptor protein or CRP

• Gene encoding this protein is crp

CAP

• Binding sites for CAP in lac, gal and ara operons all

contain the sequence TGTGA

– Sequence conservation suggests an important role in CAP binding

– Binding of CAP-cAMP complex to DNA is tight

• CAP-cAMP activated operons have very weak promoters

– Their -35 boxes are quite unlike the consensus sequence

– If these promoters were strong they could be activated even when

glucose is present

The lac control region. The activator–promoter region, just upstream of the operator, contains

the activator-binding site, or CAP-binding site, on the left (yellow) and the promoter, or

polymerase -binding site, on the right (pink). These sites have been defi ned by footprinting

experiments and by genetic analysis.

The Mechanism of CAP Action

• The CAP-cAMP complex binds to the lac promoter

– Mutants whose lac gene is not stimulated by complex had the mutation in the lac promoter

– Mapping the DNA has shown that the activator-binding site lies just upstream of the promoter

• Binding of CAP and cAMP to the activator site helps RNA polymerase form an open promoter complex

• The open promoter complex does not form, even if RNA polymerase has bound the DNA, unless the CAP-cAMP complex is also bound

• CAP plus cAMP allow formation of an open promoter complex

Proposed CAP-cAMP Activation of lac Transcription

• The CAP-cAMP dimer binds to its target site on the DNA

• The aCTD (a-carboxy terminal domain) of polymerase interacts with a

specific site on CAP

• Binding is strengthened between promoter and polymerase

• CAP-cAMP recruits polymerase to the promoter in two steps:

– Formation of the closed promoter complex

– Conversion of the closed promoter complex into the open promoter complex

• CAP-cAMP bends its target DNA by about 100° when it binds

okcKRPRPPR

B

2

Summary

• CAP-cAMP binding to the lac activator-binding site recruits RNA polymerase to the adjacent lac promoter to form a closed complex

• CAP-cAMP causes recruitment through protein-protein interaction with the aCTD of RNA polymerase

Experimental: Gel mobility shift assay

The principle of the mobility shift

assay is shown schematically. A

protein is mixed with radiolabeled

probe DNA containing a binding site

for that protein. The mixture is

resolved by acrylamide gel

electrophoresis and visualized using

autoradiography. DNA not mixed

with proteins runs as a single band

corresponding to the size of the DNA

fragment (left lane). In the mixture

with the protein, a proportion of the

DNA molecules (but not all of them at

the concentrations used) binds the

DNA molecule. Thus, in the right-

hand lane, there is a band

corresponding to free DNA, and

another corresponding to the DNA

fragment in complex with the protein.

Protein-DNA interactions ??

Experimental: Footprinting method

The stars represent the radioactive labels at

the ends of the DNA fragments, arrows

indicate sites where DNase cuts, red circles

represent Lac repressor bound to operator.

On the left, DNA molecules cut at random by

DNase are separated by size gel

electrophoresis. On the right, DNA

molecules are first bound to repressor then

subjected to Dnase treatment. The “footprint”

is indicated on the right. This corresponds to

the collection of fragments generated by

Dnase cutting at sites in free DNA, but in

DNA with repressor bound to it. In the latter

case, those sites are inaccessible because

they are within the operator sequence and

hence covered by repressor.

How to Identify the Protein-binding sites on DNA ??

7.2 The ara Operon

• The ara operon of E. coli codes for enzymes required to

metabolize the sugar arabinose

• It is another catabolite-repressible operon

Features of the ara Operon

• Two ara operators exist:

– araO1 regulates transcription of a control gene called araC

– araO2 is located far upstream of the promoter it controls (PBAD)

• The CAP-binding site is 200 bp upstream of the ara promoter,

yet CAP stimulates transcription

• This operon has another system of negative regulation

mediated by the AraC protein

The araCBAD Operon

The ara operon is also called the araCBAD

operon for its 4 genes

– Three genes, araB, A, and D, encode the

arabinose metabolizing enzymes

– These are transcribed rightward from the

promoter araPBAD

– Other gene, araC

• Encodes the control protein AraC

• Transcribed leftward from the araPc promoter

AraC Control of the ara Operon • The AraC, ara control protein, acts as both a positive and negative

regulator

• In absence of arabinose, no araBAD products are needed, AraC exerts negative control

– Binds to araO2 and araI1

– Loops out the DNA in between

– Represses the operon

• Presence of arabinose, AraC changes conformation

– It can no longer bind to araO2

– Occupies araI1 and araI2 instead

– Repression loop broken

– Operon is derepressed

Control of the ara Operon

Positive Control of the ara Operon

• Positive control is also mediated by CAP

and cAMP

• The CAP-cAMP complex attaches to its

binding site upstream of the araBAD

promoter

• DNA looping would allow CAP to contact

the polymerase and thereby stimulate its

binding to the promoter

ara Operon Summary

• The ara operon is controlled by the AraC protein

– Represses by looping out the DNA between 2 sites,

araO2 and araI1 that are 210 bp apart

• Arabinose can derepress the operon causing

AraC to loosen its attachment to araO2 and bind

to araI2

– This breaks the loop and allows transcription of operon

• CAP and cAMP stimulate transcription by binding

to a site upstream of araI

– AraC controls its own synthesis by binding to araO1

and prevents leftward transcription of the araC gene

7.3 The trp Operon Paradigm Anabolic Operon

1. Basic Features:

5 structural genes, enzymes used for Trp biosynthesis; 2 normal terminators

Control Region: Promoter, Operator, Leader sequence, Attenuator

Regulator Gene: trpR ... gene unlinked to Trp Operon ... Trp Repressor

Tryptophan is a Corepressor: Repressor-Trp complex is active Repressor

Repressor (aporepressor) by itself is inactive ... Derepression: 70-fold increase

in expression

Negative Control of the trp Operon

• Without tryptophan no trp repressor exists, just the inactive protein, aporepressor

• If aporepressor binds tryptophan, changes conformation with high affinity for trp operator

• Combine aporepressor and tryptophan to form the trp repressor

• Tryptophan is a corepressor

2. Attenuation:

additional Control in Trp operon and other aa operons ... 10-fold effect

Intrinsic transcription Terminator at the beginning of the Operon

Principle: alternative hairpin structures, one of which is active Terminator

Mechanism:

1. RNA polymerase initiates and transcribes a short coding region for a Leader peptide of 14

amino acids ... this leader is probably translated

2. Two of the 14 amino acids are Tryptophanes

3. The Attenuator region can form two alternative Hairpin structures:

Regions 1 and 2 paired, and 3 and 4 paired; or Regions 2 and 3 paired

4. Pairing 3:4 forms the transcription Terminator for Leader mRNA

Model: Presence or absence of Trp amino acid the key

In absence of Trp: ribosome stalls at the two Trp codons, permitting 2:3 pairing in Attenuator

before 4 is transcribed ... no 3:4 pairing, no transcription termination, and Trp operon is

transcribed completely

In presence of Trp: ribosome moves through the two Trp codons, disrupting 2:3 pairing in

Attenuator ... 3:4 pairing is permitted, transcription termination haripin occurs, and Trp operon is

not transcribed.

Thus: coupled transcription-translation important; termination depends on stem-loop structure

in the mRNA rather than in the DNA ... RNA polymerase pauses AFTER the stem, releases the

mRNA due to presence of string of U's pairing with DNA A's

Tryptophan (Trp) Operon: Paradigm Anabolic Operon

Tryptophan (Trp) Operon

Features of the nucleotide sequence of the trp operon

Tryptophan (Trp) Operon

Tryptophan (Trp) Operon

How transcription termination at the trp operon attenuator is controlled by the

availability of tryptophan.

Leader peptide of attenuator-controlled operons containing genes

for amino acid biosynthesis

Phe operon

His operon

7.4 Riboswitches

• Small molecules can act directly on the 5’-UTRs of mRNAs

to control their expression

• Regions of 5’-UTRs capable of altering their structures to

control gene expression in response to ligand binding are

called riboswitches

• Region that binds to the ligand is an aptamer (short RNA

sequences that bind tightly and specifi cally to ligands)

• An expression platform is another module in the riboswitch which can be: – Terminator

– Ribosome-binding site

– Another RNA element that affects gene expression

• Operates by depressing gene expression – Some work at the transcriptional level

– Others can function at the translational level

Model of Riboswitch Action

• FMN binds to aptamer in a riboswitch called the RFN element in 5’-UTR of the ribD mRNA

• Binding FMN, base pairing in riboswitch changes to create a terminator

• Transcription is attenuated

• Saves cell energy as FMN is a product of the ribD operon

Chapter 8

Major Shifts in Bacterial Transcription

Major Shifts in Bacterial Transcription

• Bacteria control the transcription of a very limited number

of genes at a time through the use of operons

• More radical shifts in gene expression require more

fundamental changes in the transcription machinery

• Three major mechanisms:

-factor switching

– RNA polymerase switching

– Antitermination : We will use the l phage to illustrate the

antitermination mechanism, and also discuss the genetic switch

used by l phage to change from one kind of infection strategy to

another.

8.3 Infection of E. coli by Phage l

• Virulent phage replicate and kill their host by lysing or breaking it open

• Temperate phage, such as l, infect cells but don’t necessarily kill

• The temperate phage have 2 paths of reproduction

– Lytic mode: infection progresses as in a virulent phage

– Lysogenic mode: phage DNA is integrated into the host genome

Lytic versus lysogenic infection by phage l

Phage l can replicate in either of two ways: lytic or lysogenic. In the lytic mode, almost all of the phage genes

are transcribed and translated, and the phage DNA is replicated, leading to production of progeny phages and

lysis of the host cells. In the lysogenic mode, the l DNA is incorporated into the host genome; after that occurs,

only one gene is expressed. The product of this gene, the l repressor, prevents transcription of all the rest of the

phage genes. However, the incorporated phage DNA (the prophage) still replicates, because it has become part

of the host DNA.

The case of phage l : layers of regulation

Lysogenic

induction (by DNA

damage): switch

from lysogenic to

lytic growth

Bacteriophage l (lambda) is a virus that infects E.coli. Upon infection

the phage can propagate in either of two ways: lytically or lysogenically

Growth Conditions:

If Growth Conditions are poor,

the Lysogenic Response is

favored.

If Growth Conditions are great,

the Lytic Response is favored.

Lysogenic Mode

• A 27-kD phage protein (l repressor, CI) appears

and binds to 2 phage operator regions

• CI (pronounced “c-one”) shuts down transcription

of all genes except for cI, gene for l repressor

itself

• When lysogeny is established the phage DNA

integrates into the bacterial genome

• A bacterium harboring integrated phage DNA is

called a lysogen and the integrated DNA is called

a prophage

• The phage DNA in the lysogen replicates along

with the host DNA

Lytic Reproduction of Phage l

• Lytic reproduction cycle of phage l has 3

phases of transcription:

– Immediate early

– Delayed early

– Late

• Genes of these phases are arranged

sequentially on the phage DNA

Genetic Map of Phage l

• DNA exists in linear form in the phage

• After infection of host begins the phage DNA circularizes

• This is possible as the linear form has sticky ends

• Gene transcription is controlled by transcriptional switches

a) The map is shown in linear form, as the DNA exists

in the phage particles; the cohesive ends (cos) are at

the ends of the map. (b) The map is shown in circular

form, as it exists in the host cell during a lytic infection

after annealing of the cohesive ends.

Lambda DNA is injected and

immediately circularizes at the

Cos cohesive ends; ligase

seals the nicks; DNA becomes

supercoiled.

Antitermination

• Antitermination is a type of transcriptional switch used by phage l

• The host RNA polymerase transcribes the immediate early genes first

• A gene product serves as antiterminator that permits RNA polymerase to ignore terminators at the end of the immediate early genes

• Same promoters are used for both immediate early and delayed early transcription

• Late genes are transcribed when another antiterminator permits transcription of the late genes from the late promoter to continue without premature termination

Antitermination and Transcription

Temporal control of transcription during

lytic infection by phage l. (a) Immediate

early transcription (red) starts at the

rightward and leftward promoters (PR and

PL, respectively) that flank

the repressor gene (cl ); transcription stops

at the rho-dependent terminators (t) after

the N and cro genes.

(b) Delayed early transcription

(blue) begins at the same promoters, but

bypasses the terminators by virtue of the N

gene product, N, which is an antiterminator.

(c) Late transcription (green) begins at a

new promoter (PR’); it would stop

short at the terminator (t) without the Q

gene product, Q, another antiterminator.

Note that O and P are protein-encoding

delayed early genes, not operator and

promoter.

N Antitermination Function

• Genetic sites surrounding the N gene include:

– Left promoter, PL

– Operator, OL

– Transcription terminator

(b) Transcription in the absence of N. RNA

polymerase (pink) begins transcribing

leftward at PL and stops at the terminator

at the end of N. The N mRNA is the only

product of this transcription.

(c) Transcription in the presence of N. N

(purple) binds to the nut region of the

transcript, and also to NusA (yellow), which,

along with other proteins not shown (NusA,

NusB, NusG, and the ribosomal S10 host

proteins), has bound to RNA polymerase.

This complex of proteins alters the

polymerase so it can read through the

terminator and continue into the delayed

early genes.

Antitermination and Q

• Antitermination in the l late region requires Q

• Q binds to the Q-binding region of the qut site as RNA polymerase is stalled just downstream of late promoter

• Binding of Q to the polymerase appears to alter the enzyme so it can ignore the terminator and transcribe the late genes

The case of phage l : Alternative patterns of gene expression

control lytic and lysogenic growth

1. CI and Cro BOTH act as Repressors, each binding to two operators OL and

OR, adjacent to the promoters PL and PR.

2. These operators are each composed of three binding sites for Cro and CI,

both the binding specificities of Cro and CI to these three sites are reversed.

Binding specificity for CI: OR3 < OR2 < OR1 and OL1 > OL2 > OL3

Binding specificity for Cro: OR3 > OR2 ~ OR1 and OL1 ~ OL2 < OL3

A monomer of l repressor,

indicating various surfaces

involved in different activities

carried out by the protein.

“tetramerization” denotes the

region where two dimers interact

when binding cooperatively to

adjacent sites on DNA.

l repressor binds to operator sites cooperatively

The l repressor monomers interact

to form dimers, and those dimers

interact to form tetramers. These

interactions ensure that binding of

repressor to DNA is cooperative.

Concentration, affinity, and

cooperative binding regulate the

transcription

When CI (λ repressor) binds to OR1, it prevents transcription from PR. Thus, CI shuts off Cro

and all rightward gene expression. This includes Q gene expression, which in turn is required

for Late gene expression.

When CI binds to OL1, it prevents transcription from PL. Thus, it shuts off N protein

expression, which in turn is required for Delayed Early gene expression.

Each of these actions of CI tends to turn off the Lytic Response.

When CI also binds to OR2, it activates transcription from the PRM promoter (Promoter for

Repressor Maintenance). Transcription proceeds leftward, through the CI genes. Thus, CI

positively controls its own expression. This is an example of autoregulation. ...

At higher CI concentrations, CI binds to OR3; this now prevents expression from the PRM

promoter. CI expression thus is tightly controlled by CI itself via transcription from the PRM

promoter and binding of CI to the OR2 and OR3 sites.

Each of these actions of CI tends to turn on the Lysogenic Response.

Cro:

when Cro binds to OR3, it prevents transcription from PRM. Thus, Cro shuts off CI gene

expression, thereby preventing the Lysogenic response.

At higher Cro concentrations, Cro binds to OR1 and OL1, preventing gene expression from PR

and PL. Thus, Cro turns off its own gene expression (autoregulation). However, by this

time, the Q gene has been expressed, Late genes are being expressed, and the Lytic Cycle

is well underway.

l Repressor and Cro bind in different patterns to control lytic and

lysogenic growth

l Repressor (CI) and Cro bind in different patterns to control lytic

and lysogenic growth

Lysogenic induction requires proteolytic cleavage of l repressor

DNA damage >> activating SOS response system >> activating RecA

protein>> stimulate autocleavage of LexA

l repressor (CI) has evolved to resemble LexA, ensuring that l repressor

too undergoes autocleavage in response to activated RecA.

l repressor cleavage >> induction of lytic pathway

l repressor: positive autoregulation and negative autoregulation

· A repressor monomer has two distinct

domains.

· The N-terminal domain contains the DNA-

binding site.

· The C-terminal domain dimerizes.

· Binding to the operator requires the

dimeric form so that two DNA-binding domains

can contact the operator simultaneously.

· Cleavage of the repressor between the

two domains reduces the affinity for the operator

and induces a lytic cycle.

The l lytic cascade is interlocked with the circuitry for lysogeny

Lambda immediate early and

delayed early genes are needed for

both lysogeny and the lytic cycle

· Lambda has two immediate

early genes, N and cro, which are

transcribed by host RNA polymerase.

· N is required to express the

delayed early genes.

· Three of the delayed early

genes are regulators.

· Lysogeny requires the delayed

early genes cII-cIII.

· The lytic cycle requires the

immediate early gene cro and the

delayed early gene Q.

The lytic cycle depends on antitermination

· pN is an antitermination factor that allows RNA polymerase to continue

transcription past the ends of the two immediate early genes.

· pQ is the product of a delayed early gene and is an antiterminator that allows

RNA polymerase to transcribe the late genes.

· Because lambda DNA circularizes after infection, the late genes form a single

transcription unit.

Lysogeny is maintained by an

autogenous circuit. If this

circuit is interrupted, the lytic

cycle starts

· Repressor binding at OL blocks

transcription of gene N from PL.

· Repressor binding at OR blocks

transcription of cro but also is required for

transcription of cI.

· Repressor binding to the operators

therefore simultaneously blocks entry to

the lytic cycle and promotes its own

synthesis.

· Mutants in the cI gene cannot

maintain lysogeny.

· cI codes for a repressor protein that

acts at the OL and OR operators to block

transcription of the immediate early

genes.

Because the immediate early genes

trigger a regulatory cascade, their

repression prevents the lytic cycle from

proceeding.

The cII and cIII genes are needed to establish lysogeny

· The delayed early

gene products cII and cIII

are necessary for RNA

polymerase to initiate

transcription at the

promoter PRE.

· cII acts directly at

the promoter and cIII

protects cII from

degradation.

· Transcription from

PRE leads to synthesis of

repressor and also blocks

the transcription of cro.

Lysogeny requires several events

· cII/cIII cause repressor

synthesis to be established and also

trigger inhibition of late gene

transcription.

· Establishment of repressor

turns off immediate and delayed

early gene expression.

· Repressor turns on the

maintenance circuit for its own

synthesis.

· Lambda DNA is integrated into

the bacterial genome at the final

stage in establishing lysogeny.

The cro repressor is needed for lytic infection

· Cro binds to the same

operators as repressor but with

different affinities.

· When Cro binds to OR3, it

prevents RNA polymerase from

binding to PRM, and blocks

maintenance of repressor.

· When Cro binds to other

operators at OR or OL, it prevents

RNA polymerase from expressing

immediate early genes, which

(indirectly) blocks repressor

establishment.

Determining the Fate of a l Infection

• Balance between lysis or lysogeny is delicate

• Place phage particles on bacterial lawn

– If lytic infection occurs

• Progeny spread and infect other cells

• Circular hole seen in lawn is called plaque

– Infection 100% lytic gives clear plaque

– Plaques of l are usually turbid meaning live

lysogen is present

• Some infected cells suffer the lytic cycle, others

are lysogenized

Battle Between cI and cro

• The cI gene codes for

repressor, blocks OR1, OR2,

OL1, and OL2 so turning off

early transcription

• This leads to lysogeny

• The cro gene codes for Cro

that blocks OR3 and OL3,

turning off transcription

• This leads to lytic infection

• Gene product in high

concentration first determines

cell fate

Lysogen Induction

• When lysogen suffers DNA

damage, SOS response is

induced

• Initial event is seen in a

coprotease activity in RecA

protein

• Repressors are caused to cut

in half, removing them from l

operators

• Lytic cycle is induced

• Progeny phage can escape

potentially lethal damage

occurring in host

What determines the balance between

lysogeny and the lytic cycle?

· The delayed early stage when both Cro

and repressor are being expressed is

common to lysogeny and the lytic cycle.

· The critical event is whether cII causes

sufficient synthesis of repressor to

overcome the action of Cro.

High levels of cII favor lysogeny, while low

levels favor the lytic pathway. The instability

of the cII protein is due to degradation by

proteases.

Under favorable growth conditions,

proteases are plentiful, the cII protein is

degraded, and, hence, the lysogenic cycle is

not activated. Under poor growth

conditions, proteases are less abundant and

there is less degradation of cII, so those

promoters are activated and lysogeny is

favored.