Regulatory Circuits (Lectures 12-14)

What is enzyme induction and why is it beneficial?

It is the process in which a bacterial cell only makes enzymes needed for growth on a particular substrate in the presence of the molecule / nutrient. This ensures efficient systems (not wasting energy on enzymes that are not necessary).

Promoter

binds RNA Polymerase and initiates transcription (production of mRNA)

Operator

binds regulatory proteins to alter transcription

Negative regulation

when gene expression is turned off by a regulatory protein (a repressor)

Repressor

regulatory protein that turns gene expression off

Activator

regulatory protein that turns gene expression on

Positive regulation

when gene expression is turned on by a regulatory protein (an activator)

How are gene expression levels when an activator protein is inactive?

no or low

How are gene expression levels when a repressor protein is inactive?

constitutive (normal)

What regulatory style controls the lac operon?

negative regulation; controlled by a repressor protein

What molecule must be present to start the transcription of the lac operon?

lactose

What is the general path of transcription of the lac operon?

-Lactose binds allosterically to the repressor protein, which causes a conformational change.

-The repressor protein then detaches from the operator region.

-This allows RNA Polymerase to transcribe the genes in the lac operon, producing the enzymes B-galactosidase, permease, and transacetylase

What are the three enzymes produced in the lac operon?

-B-galactosidase

-Permease

-Transacetylase

Who were the 3 scientists that won a Nobel prize in 1965 for finding genetic regulatory mechanisms for enzyme and virus synthesis?

-Francois Jacob

-Jacques Monod

-Andre Lwoff

What were the 7 big discoveries of Jacob, Monod, and Lwoff?

1. Inducers regulate the synthesis of new B-gal

2. Inducers differ from substrates

3. Genes on an operon are controlled together

4. Lacl is a repressor

5. Lacl+ is trans-dominant over lacl-

6. Lacl-S is dominant and cannot bind the inducer

7. Operator-constitutive (O-c) mutants are cis-dominant

Describe the discovery that inducers regulate new B-gal synthesis

-Originally, people thought that inducers led to increased B-gal being activated, but that the enzyme itself was pre-existing in the cell.

To test this, the researchers added radioactive amnio acids either before or after induction, and found that radioactivity accumulated in the B-gal enzyme.

This meant that induction led to new and fast synthesis.

They also found that if the inducer was removed, synthesis of B-gal would stop. This showed that B-gal is synthesized in the presence of an inducer, not simply activated.

Described the discovery that inducers differ from substrates

In the lac operon, lactose (or allolactose) is both and inducer AND a substrate. However, other related molecules that are NOT substrates can ALSO act as inducers. For example, the “mimic” IPTG can induce the operon.

So, the component of the operon that recognizes the inducer is distinct from the enzyme.

Describe the discovery that genes on the lac operon are controlled together

When permease (one of the enzymes produced by the operon) production is induced, B-gal production is co-induced. While mutations in these 2 genes showed that they were separate, gene mapping shows that lacZ, lacY, and lacA are very closely linked.

This led to the concept of the operon and mRNA, which was later verified experimentally.

What was the PaJaMo experiment?

Originally, people thought that the Lacl- allele was making an internal inducer because lacl- was always ON. If this were the case, lacl- would be dominant over lacl+.

After testing, however, they found that the lacl+ allele could actually repress B-gal synthesis in the presence of lacl-. This meant the wild type (lacl+) allele was dominant over the lacl-, meaning that the product of this gene was actually a repressor and that the inducer “inhibits the inhibitor”.

Describe the discovery that lacl is a repressor (not an inducer)

-Mutated lacl genes (lacl-) resulted in a constitutive, “always on” production of enzymes

-These mutated genes did not respond to an inducer and were the first regulatory mutant

-Lacl was mapped close to lacZYA

-PaJaMo Experiment: showed that lacl was a repressor and inducer molecules “inhibit the inhibitor”; lacl+ dominant over lacl-

Describe the discovery that lac+ is trans-dominant over lacl-

After performing complementation studies using F’ factors, researchers found that the lacl gene encoded a “diffusible factor” (repressor) that was initially absent from the cytoplasm of the recipient cell. If there is one lacl+ gene and one lacl- gene, there will still be repressor molecules being produced.

Describe the discovery that Lacl-S mutants are dominant and cannot bind the inducer

-Lacl-S mutants did not express the genes lacZ, lacY, or lacA experimentally

-The mutation was mapped to the Lacl gene

-Mutation acts as a “super repressor”

-Lacl-S is trans-dominant over lacl+ AND lacl-

-This mutation prevents the repressor from binding to allolactose (the inducer), meaning the repressor will always bind the operator and the operon will be non-inducible

What is an O-c mutant?

A mutant where the repressor cannot bind to the altered operator, so expression of the lac operon is always ON, even in the absence of an inducer

Describe the discovery of Operator-constitutive mutants

Originally, scientists hypothesized that the repressor protein interacted with an “operator” near the beginning of the genes it controlled. To test this, they first looked for constitutive mutants in cells containing 2 copies of the repressor (to limit the possibility that the normal repressor would mutate).

These constitutive mutants had a mutation in the operator region, and were cis-dominant over lacl+ and lacl-S (operators are cis-acting because they can only act on the regions near them).

These cells had expression always turned ON because the repressor cannot bind to the altered operator region.

6 Key Concepts of Lac Operon

1. inducers regulate new B-gal synthesis

2. inducers and substrates are different

3. Genes are controlled together (operonic)

4. Lacl encodes a diffusible repressor (not an activator)

5. Inducers interact directly with the repressor protein

6. Repressor protein interacts with the operator

What techniques were used by Jacob and Monod to deduce the “operon model” in 1961?

-biochem

-mutant isolation

-gene mapping

-complementation analysis (dominant vs recessive alleles)

-Trans vs cis affects

-Deductive logic

Phage lambda

-infects E. Coli

-Siphpoviridae

-Double-stranded DNA genome

-Long, flexible tail with an icosahedral head

Lytic

phage lifecycle resulting in lysis of bacterial cell upon release of progeny phage

Lysogenic

phage lifecycle resulting in stable carriage of the phage (prophage) within the host cell (lysogen)

Virulent phage

can only replicate via the lytic cycle

Temperate phage

can replicate with either the lytic or lysogenic cycle (like phage lambda)

Lysogen

host cell harboring prophage during lysogeny

Prophage

latent form of temperate phage that stays within the lysogen because it has integrated into the host chromosome

What are the 4 major / important events in the phage lambda lifecycle?

1. Infection

2. Decision — Lytic or lysogenic?

3. Maintenance (if lysogenic)

4. Induction — “switch” to lytic eventually (if lysogenic)

Step 1: Infection

-The phage genome is linear in the phage head

-After injection, the lambda genome circularizes in the host

-This process and modularly organized and aided by cohesive end sites known as “cos sites”

Steps of lytic cycle: phage assembly and release

1. Head and tail proteins synthesized

2. DNA packaged into phage heads

3. Tails added on

4. Host lysed and new phages released

Steps of Lysogenic Cycle: Phage integration and maintenance

1. Lambda integrates into host genome

2. Prophage stably maintained

3. Prophage passed to daughter cells

Between what 2 sites does integration of lambda DNA into the host chromosome occur?

attP (attachment on Phage) and attB (attachment on Bacteria)

What enzyme is needed for site-specific recombination during lysogeny (for lambda to be linearized into host chromosome)?

Int (integrase)

When is it a good time to make more phages?

when there is high bacterial growth and lots of food/energy available

When is it a good time for the phage to integrate into the genome?

if the cell is unlikely to have resources available to produce many phages

When is a good time for a phage to leave the lysogenic cycle and enter the lytic phase?

if the cell is damaged and likely to die

What 2 promoters are transcribed by the host RNA Polymerase right after injection?

PL (produces N) and PR (produces Cro)

What does the Cro gene do?

encodes Cro protein; expressed early in infection and plays a major role in establishing lytic growth

What does N do?

encodes for an anti-terminator protein, which allows for transcription past 2 terminators and leads to early gene expression

What is NUT?

N utilization site

Cro

DNA-binding protein that represses transcription; promotes lytic cycle

CII

DNA-binding protein that activates transcription; promotes lysogenic cycle

CI

DNA-binding protein that can active or repress transcription

-activates its own expression

-represses genes required for the lytic cycle

-maintains lysogeny

How is the “decision” to switch from lysogenic to lytic made?

CII is the deciding protein! CII promotes the lysogenic phase while Cro enables the lytic. Normally, the host cell produces proteases that degrade CII. In cells that are healthy and actively growing (meaning they will have the resources to make phage parts), proteases are produced in abundance and CII is degraded. This means Cro protein wins and the lytic cycle proceeds.

However, when a host cell is starved, they make less protease. Thus, CII is not degraded to the same extent and the CII proteins stay intact and stable. CII “wins” over Cro and activates the lysogenic cycle.

What are the key proteins and their roles in the late lytic cycle?

CII degraded by proteases

Cro represses expression of cl and of early genes

Cro eventually represses its own expression and represses the replication genes

Q acts as an anti-terminator, enabling expression of the late lytic genes that code for the tail, head, and cell lysis

When CII is not degraded in the late lysogenic cycle, what is its role?

it works as an activator protein to turn on…

1. int gene, which causes integration of lambda into the host chromosome

2. CI repressor, which activates its own expression and binds to O-L and O-R to repress the other phage genes

3. P-RE, which is a promoter for repressor establishment

Role of CI in lysogeny

-maintains prophage

-acts as a repressor of all phage genes, but an activator of itself

-keeps the phage genome “silent” in bacterial chromosome until induction

Key proteins to keep a stable prophage

1. CII promotes int and cl genes

2. Integration of DNA via Integrase

3. CI maintains prophage

Summary of steps / proteins in phage infection

1. Host RNA Polymerase transcribes cro and N genes

2. Anti-terminator protein N enables transcription of left and right genes

3. If resources and proteases abundant (LYTIC)

   1. CII degraded

   2. Cro represses cl

   3. Q anti-terminates and allows late gene activation

   4. Lytic cycle proceeds

4. If resources and proteases NOT abundant (LYSOGENIC)

   1. CII remains active

   2. cl transcription proceeds at a high level

   3. Integrase (Int) protein integrates lambda into the host chromosome

   4. CI shuts off all other phage genes

CI

repressor; maintains lambda as prophage in lysogenic state by repressing main promoters and activating its own expression

Cro

expressed early in infection; plays a major role in establishing lytic growth

CII

major activator protein in deciding lytic or lysogenic fate after infection; levels affected by host proteases

CIII

protects CII from degradation by proteases

N

anti-terminator; enables transcription past certain terminators resulting in early gene expression

Q

anti-terminator; enables expression of late lytic genes (head, tail, lysis genes)

Int

promotes integration of the lambda genome into the host chromosome for establishment of lysogeny (between attP and attB sites)

Xis

works with Int to excise lambda from host chromosome during induction to lytic growth (between attP and attB sites)

How did they discover some of the genes that control the lytic vs lysogenic pathways?

-Lambda phage forms turbid plaques because lysogens are immune to further infections (so there will be growth in the plaques)

-In some cases, the plaques were clear, meaning there was no growth and therefore no lysogeny

• These plaques contained mutants named cI, cII, and cIII after “clear mutants”

How does UV light lead to a switch to lytic growth?

UV radiation inactivates CI, allowing Cro to be synthesized and lytic growth to occur

What are the 3 basic components of the “switch”?

• Regulatory proteins: CI (repressor) and Cro

• Operator sites on the phage DNA and their promoters

• RNA Polymerase (essential for transcription)

What are the 2 positions of the “switch” and how does each position effect the transcription of CI and Cro?

1. Lysogeny: CI on, Cro off (maintenance)

2. Lytic: CI off, Cro on (induction)

The genes cl and cro are transcribed ______________

divergently

What are the two types of sites located in the region between the cl and cro genes?

1. Operator (binds CI and Cro)

2. Promoter (binds RNA Polymerase)

How many operator sites are there for CI and Cro?

3

How do the operator sites overlap the promoters?

OR1 and OR3 overlap 1 promoter while OR2 overlaps both promoters

What are the 2 promoters for RNA Polymerase in the switch?

P-RM and P-R

Do the 2 promoters in the switch overlap?

No

Protein binding to DNA is ____________

reversible; some regulator binds and some falls off

• affinities can determine the order of binding relative to protein concentrations

Role of RNA Polymerase / Key Features in the Switch

-Transcribes DNA into mRNA

-Provided by the host, not the phage

-Binds either P-R or P-RM, but NEVER BOTH

Which switch promoter needs a regulatory protein?

P-RM — needs CI as an activator

CI dimers

-majority of CI is dimeric in lysogenic cell

-interactions at C-terminal

-dimers use N-terminal domain to bind DNA

-Each OR can bind 1 CI dimer along 1 side of the DNA helix

How does CI work for NEGATIVE control?

at OR2, it turns off cro gene by preventing RNA Polymerase from binding to the cro promoter (PROMOTER OCCLUSION)

How does CI work for POSITIVE control?

at OR2, it helps the RNA Polymerase bind and begin transcription of the cl gene (upregulation) — positive activation

How does CI increase RNA Polymerase affinity for P-RM?

by providing protein-protein interactions in addition to protein-DNA interactions

What are the two key molecular functions of CI in the switch?

promoter occlusion and positive activation

If CI is bound to OR1…

both cl and cro are turned off, there is no activation and no occlusion

If CI is bound to OR2…

cl is turned on (activation) but cro is off (exclusion)

-lysogenic maintenance

If CI is bound at OR3…

cl turned off (RNA Polymerase exclusion), but cro is turned on

-Lytic induction

Which ORs are usually bound by CI in lambda lysogens?

OR1 and OR2, results in cl on and cro off

What 2 factors contribute to CI binding?

1. intrinsic affinity

2. cooperativity

Describe the cooperativity behavior of CI

-bound CI at OR1 increases affinity for CI at OR2 with protein-protein interactions

-binding at OR3 is weak because there is no cooperativity (since CI C-terminal domains are unavailable)

-dimers of CI “lean” toward each other

-CI binding at OR2 can still bind RNA Polymerase because it interacts with the N-terminus

What does the switch look like during lysogeny maintenance?

-CI bound at OR1 and OR2

-cro inhibited

-cl expressed

-very stable

-state inherited by daughter cells (epigenetic)

What is the role of RecA?

it senses DNA damage caused by UV radiation and becomes a co-protease. As a co-protease, it cleaves CI into 2 domains and results in non-functional CI. This clears the operator sites of CI and changes the switch to induce the lytic phase.

Results of CI cleavage by RecA

1. Activation of P-RM lost, so cl is lost

2. Repression of excision genes lost, leading to decreased cl

3. RNA Polymerase binds P-R and now transcribes cro genes

4. Cro now determines the course of events

   1. prophage excises from host chromosome

   2. lytic growth

How does Cro interact / bind with the switch?

-Almost all Cro is dimeric

-Dimers can bind to each OR site in the absence of CI

-Binds on one side of the DNA helix

-Binds same OR sites as CI, but with OPPOSITE EFFECTS

Effects of Cro on switch

-binds each operator site independently (no cooperativity)

-only a negative regulator

-effects are roughly opposite that of CI

Steps of Switch during Induction

-CI removed from operators

-Cro bound to OR3 stops any possible CI expression

-Expression from P-R allows expression of phage lytic genes

-Cro essentially shuts down all cl expression and the late lytic gene expression can happen (making heads and tails)

-Eventually, cro shuts off its own transcription and that of early phage lytic genes

How / when is the prophage excised from the host?

-after CI cleavage, you lose repression of P-Int (promoter for Integrase)

-Int and Xis proteins are made

-Together, Xis/Int promote excision of lambda from the host