BIOL 3301 Ch 14: Gene Regulation in Bacteria

gene regulation

• level of gene expression can vary under different conditions

constitutive genes

• unregulated genes

• have essentially constant levels of expression

• frequently code for proteins that are continuously necessary for the survival of the organism

  • (it’s fine that they’re unregulated bc the benefit of regulating genes is that coded proteins will be produced only when required)

only when required

• the benefit of regulating genes is that coded proteins will be produced ?

constitutive genes

• gene expression always “on”

• unregulated

regulated genes

• gene expression sometimes “on”

ch 14 bacteria vs ch 15 eukarotes

• how do proteins that bind to DNA regulatory sequences somehow manage to either increase or decrease the rate of transcription by RNA polymerase?

  • ?

    • Transcription factors control gene expression.

    • Repressors block transcription (off).

    • Activators help transcription (on).

    • Example: Lac operon regulates gene.

  • ?

    • DNA is inside the nucleus.

    • Enhancers increase transcription (help).

    • Silencers decrease transcription (block).

    • Additional proteins fine-tune regulation.

gene regulation

• ? is important for these cellular processes

  • metabolism

  • response to environmental stress

  • cell division

• regulation can occur at ANY of the points on the pathway to gene expression

  • there are conditions under which some genes are expressed and other conditions under which they are not

common gene expression regulation points in bacteria

• transcription (gene→mRNA)

  • genetic regulatory proteins bind DNA and control transcription rate

  • in attenuation, transcription is terminated soon after it starts due to formation of transcriptional terminator

• translation (mRNA→ protein)

  • translational repressor proteins bind mRNA and prevent translation from starting

  • riboswitches produce an mRNA conformation preventing translation from starting

  • antisense RNA binds mRNA and prevents translation from starting

• posttranslation (protein→ functional protein)

  • in feedback inhibition, the product of a metabolic pathway inhibits the first enzyme in the pathway

  • covalent modification to the structure of a protein can alter its function

transcription gene regulation in bacteria

• genetic regulatory proteins bind to the DNA and control the transcription rate

  • Lac Operon: The lac repressor blocks transcription when lactose is absent. When lactose is present, it binds to the repressor, allowing RNA polymerase to transcribe the genes needed for lactose metabolism.

  • Trp Operon: The trp repressor binds to the operator region and prevents transcription when tryptophan is abundant. When tryptophan is scarce, the repressor detaches, allowing transcription for tryptophan biosynthesis.

• occurs when gene is being copied into mRNA

posstranslational gene regulation in bacteria

• in feedback inhibition, the product (final) of a metabolic pathway inhibits the first enzyme pathway

  • prevents the pathway from producing more of the product than needed

• covalent modification to the structure of a protein can alter its function

  • e.g., phosphorylation, acetylation, methylation, ubiquitination,

  • modifications change protein’s shape, activity, stability or localization

    • ex., phosphorylation can activate/deactivate enzymes by altering their conformation

    • ubiquitination may mark some proteins for degradation

• regulation can occur as protein is undergoing changes to become a functional protein

transcription initiation

• the most common way to regulate gene expression in bacteria

  • the rate of RNA synthesis can be increased or decreased

• involves regulatory transcription factors (RTFs)← proteins!

  • repressors: bind DNA and inhibit transcription

  • activators: bind DNA and increase transcription

regulatory transcription factors RTFs

• proteins that regulate transcription

• repressors: bind DNA and inhibit transcription

• activators: bind DNA and increase transcription

repressors

• regulatory transcription factors RTFs that bind DNA and inhibit transcription

activators

• regulatory transcription factors RTFs that bind DNA and increase transcription

negative control

• transcriptional regulation by repressor proteins

  • gene expression on→ (repressor protein action)→ off

positive control

• transcriptional regulation by activator proteins

  • gene expression: off → (activator protein action)→ on

small effector molecules

• ? affect transcription regulation

  • bind to regulatory transcription factors but not to DNA directly

• may increase or decrease transcription

  • inducers

    • increase transcription

    • bind activators and cause them to bind DNA

    • bind to repressors and prevent them from binding to DNA

    • genes regulated in this manner are inducible

  • may inhibit transcription

    • corepressors bind repressors and cause them to bind DNA

    • inhibitors bind activators and prevent them from binding DNA

    • genes regulated in this manner are repressible

inducers

• small effector molecule that increases transcription

  • bind to regulatory transcription factors but not DNA directly

    • bind activators and cause them to bind DNA

    • bind repressors and prevent them from binding to DNA

• genes regulated in this manner are inducible

inducible genes

• regulated by small effector molecules (inducers)

  • inducers increase transcription

    • bind to regulatory transcription factors but not DNA directly

      • bind activators and cause them to bind DNA

      • bind repressors and prevent them from binding to DNA

corepressors

• bind repressors and cause them to bind to DNA

• small effector molecule that inhibits transcription

• regulate repressible genes

inhibitors

• bind activators and prevent them from binding DNA

• small effector molecule that inhibits transcription

• regulate repressible genes

repressible genes

• regulated by small effector molecules that inhibit transcription

  • corepressors bind repressors and cause them to bind DNA

  • inhibitors bind activators and prevent them from binding DNA

repressor protein, inducer molecule, inducible gene

• no inducer present

  • repressor protein blocks transcription

• inducer present

  • inducer binds repressive to repressor protein, causing a conformation change that prevents the repressor from binding to the DNA, thereby allowing transcription to proceed

activator protein, inducer, inducible gene

• no inducer present

  • activator protein can’t bind DNA, transcription does not occur

• inducer present

  • inducer binds activator protein, allowing it to bind to the DNA and activate transcription

repressor protein, corepressor molecule, repressible gene

• no corepressor present

  • repressor protein won’t bind DNA→ transcription occurs

• corepressor present

  • corepressor binds repressor protein, causing a conformation change that allows the repressor to bind the DNA→ inhibit transcription

activator protein, inhibitor molecule, repressible gene

• no inhibitor present

  • activator protein binds DNA without needing an effector molecule→ transcription proceeds

• inhibitor present

  • inhibitor binds to activator protein, causing a conformational change that prevents the activator from binding to the DNA→ transcription inhibited

enzyme adaptation

• a particular enzyme appears within a living cell only after the cell has been exposed to the substrate for that enzyme

lactose metabolism

• in 20th century, Francois Jacob and Jacques Monod at Pasteur Institute in Paris were interested in the phenomenon of enzyme adaptation

  • particular enzyme appears in cell only after cell has been exposed to enzyme’s substrate

• their focus was on ? in E. coli

  • observed

    • medium w/o lactose

      • no detectable presence of beta-galactosidase in E. coli cell

    • medium w/ lactose

      • lots of beta-galactosidase in E. coli cell

  • why?

    • seemed like the cell could "detect" the presence of lactose (its substrate) and began to produce the enzyme (beta galactosidase) in response.

    • However, lactose isn't the cell's "favorite food," so the cell produces the enzyme only when lactose is present, indicating inducible gene regulation

wo lactose

• E coli grown in ? medium

• does not produce the enzyme β-galactosidase (an enzyme needed to metabolize lactose)

  • seemed like the cell could "detect" the presence of lactose (its substrate) and began to produce the enzyme (beta galactosidase) in response.

  • However, lactose isn't the cell's "favorite food," so the cell produces the enzyme only when lactose is present, indicating inducible gene regulation

w lactose

• E coli grown in ? medium

• cell starts producing large amounts of β-galactosidase.

  • a

operon

• regulatory unit consisting of a few protein-coding genes under control of 1 promoter

• codes a polycistronic mRNA

  • contains coding sequence for 2 or more protein-coding genes

• allows bacterium to coordinately regulate a group of genes that code proteins with a common functional goal

• E. coli genes involved in lactose utilization have 2 transcriptional uniits

  • Lac Operon: One transcriptional unit that includes the genes responsible for lactose metabolism (e.g., lacZ, lacY, lacA).

  • lacI Gene: A separate transcriptional unit that codes for the lac repressor protein, which controls the lac operon.

2 transcriptional units

• genes in E coli involved in lactose utilization have ?

  • lac operon

    • One transcriptional unit that includes the genes responsible for lactose metabolism (e.g., lacZ, lacY, lacA)

    • regulatory unit that codes for proteins involved in lactose metabolism

    • DNA elements that control transcription

      • promoter→ bind RNA polymerase

      • operator→ bind lac repressor protein

      • CAP site→ binds the catabolite activator protein CAP

      • terminator→ ends transcription

    • protein coding genes

      • lacZ

        • codes beta galctosidase

        • enzymatically cleaves lactose and lactose analogs

        • also converts lactose→ allolactose (an isomer)

      • lacY

        • codes lactose permease

        • membrane protein required for transport of lactose and analogues

      • lacA

        • codes galactosidase transacetylase

        • covalently modifies lactose and analogues

        • prevents toxic buildup of nonmetabolizable lactose analogs

  • lacl gene

    • A separate transcriptional unit that codes for the lac repressor protein, which controls the lac operon.

    • not considered part of lac operon

    • has its own promoter, i promoter

    • constitutively expressed at fairly low intervals

    • codes lac repressor

      • lac repressor protein fxs as a tetramer

    • only a small amount of protein is needed to repress the lac operon

lac operon

• One transcriptional unit that includes the genes responsible for lactose metabolism (e.g., lacZ, lacY, lacA) in E coli

• regulatory unit that codes for proteins involved in lactose metabolism

• (its actual parts are) DNA elements that control transcription

  • promoter→ bind RNA polymerase

  • operator→ bind lac repressor protein

  • CAP site→ binds the catabolite activator protein CAP

  • terminator→ ends transcription

• protein coding genes

  • lacZ

    • codes beta galctosidase

    • enzymatically cleaves lactose and lactose analogs

    • also converts lactose→ allolactose (an isomer)

  • lacY

    • codes lactose permease

    • membrane protein required for transport of lactose and analogues

  • lacA

    • codes galactosidase transacetylase

    • covalently modifies lactose and analogues

    • prevents toxic buildup of nonmetabolizable lactose analogs

lac promoter

• bind RNA polymerase

• part of lac operon

operator site laco

• sequences of bases that bind lac repressor protein, which blocks transcription when lactose is absent.

• part of lac operon

CAP site

• DNA sequence recognized by/binds an activator protein the catabolite activator protein CAP

• part of lac operon

lac terminator

• ends transcription

• part of lac operon

lac z

• codes beta galactosidase

• enzymatically cleaves lactose and lactose analogs

• also converts lactose→ allolactose (an isomer)

• part of lac ooperon

lacY

• codes lactose permease

• membrane protein required for transport of lactose and analogues

• part of lac operon

lacA

• codes galactosidase transacetylase

• covalently modifies lactose and analogues

• prevents toxic buildup of nonmetabolizable lactose analogs

• part of lac operon

lacl gene

• A separate transcriptional unit that codes for the lac repressor protein, which controls the lac operon.

• not considered part of lac operon

• has its own promoter, i promoter

• constitutively expressed at fairly low intervals

• codes lac repressor

  • lac repressor protein fxs as a tetramer

• only a small amount of protein is needed to repress the lac operon

DNA sequence organization in lac region of E coli chromosome

• i Promoter: Drives transcription of the lacI gene (codes for the lac repressor).

• CAP Site: Binds the catabolite activator protein (CAP) to help activate the operon when glucose is low.

• lac Promoter (lacP): Controls the transcription of the lacZ, lacY, and lacA genes as a single polycistronic mRNA.

• Operator Site (lacO): Binds the lac repressor protein, which blocks transcription when lactose is absent.

• lac Terminator: Marks the end of transcription for the lac operon.

• lacZ: Codes for β-galactosidase, which breaks down lactose into glucose and galactose.

• lacY: Codes for lactose permease, which allows lactose entry into the cell.

• lacA: Codes for galactoside transacetylase, involved in detoxifying certain sugars.

protein fx in lactose metabolism

• lactose permease

  • facilitates active transport of lactose→ cytoplasm

  • use proton gradient to co-transport lactose and hydrogen ions (H+)

• β-Galactosidase

  • break down lactose→ glucose + galactose

  • catalyzes a side rxn, converting lactose→ allolactose (an inducer of the lac operon)

    • allolactose is further broken down→ glucose and galactose via β-galactosidase.

lactose permease

• protein

• facilitates active transport of lactose→ cytoplasm

• use proton gradient to co-transport lactose and hydrogen ions (H+)

beta galactosidase

• break down lactose→ glucose + galactose

• catalyzes a side rxn, converting lactose→ allolactose (an inducer of the lac operon)

  • allolactose is further broken down→ glucose and galactose via β-galactosidase.

• protein

lac operon

lac operon

• ? can be transcriptionally regulated

  • by repressor protein (negative control)

  • by activator protein (positive control)

• Inducible, negative control mechanism

  • lac repressor protein

  • inducer is allolactose (regulation)

    • acts like an inducer that inactivates the lac repressor via allosteric regulation

  • In absence of lactose in environment

    • inducer allolactose is also absent

    • lac repressor/repressor protein is tightly bound to the operator site (lacO), preventing RNA polymerase from transcribing the lac operon

  • when lactose is present in environment

    • inducer allolactose is available

      • lactose → (β-galactosidase)→ allolactose

    • allolactose binds repressor, altering the conformation of the repressor protein, which prevents i from binding the operator site (lacO)

    • this allows RNA polymerase to transcribe the lac operon genes, enabling lactose metabolism

• cycle of induction and repression

  • ensures lac operon is only active when lactose is available, allowing efficient regulation of lactose metabolism in E.coli

lactose absent

• inducer allolactose is also absent

• lac repressor/repressor protein is tightly bound to the operator site (lacO), preventing RNA polymerase from transcribing the lac operon

lactose present

• inducer allolactose is available

  • lactose → (β-galactosidase)→ allolactose

• allolactose binds repressor, altering the conformation of the repressor protein, which prevents it from binding the operator site (lacO)

• this allows RNA polymerase to transcribe the lac operon genes, enabling lactose metabolism

induction and repression cycle

• lactose available

  • small amount lactose→ (via β-galactosidase) → allolactose

  • allolactose binds repressor, causing it to fall of the operator site (change conformation) which prevents it from binding the operator site (lacO)

  • RNA polymerase transcribes lac operation genes

  • lac operon proteins are synthesized

    • promotes efficient uptake and lactose metabolism

      • lactose is depleted (lactose becomes more unavailable)

      • allolactose levels decrease

      • allolactose released from repressor, allowing it to bind to the operator site

      • more proteins involved with lactose utilization are degraded

        • lactose permease

        • beta galactosidase

        • lac repressor

        • galactoside transacetylase

• purpose

  • ensures lac operon is only active when lactose is available, allowing efficient regulation of lactose metabolism in E.coli

lacl gene

• In 1950s, Jacob, Monod, and Pardee ID’ed mutant strains in bacteria with abnormal lactose adaptation

• lacl gene mutation: (lacl-)

  • defect in the lacl gene

  • led to constitutive expression of lac operon, even when lactose was absent

    • basically the continuous expression of the lac operon→ always producing the enzymes needed for lactose metabolism

  • these mutations were mapped very close to the lac operon on the bacterial chromosome

• bacterial conjugation

  • researchers used bacterial conjugation to introduce different portions of the lac operon onto different strains

  • F’ factors (plasmids) were used to transfer parts of the lac operon, such as the lacl gene, into recipient bacteria

• merozygotes (partial diploids)

  • after transferring the lacl gene via F’ factors, bacteria could have 2 copies of the lacl gene: one on the chromosome and one on the plasmid (F’ factor)

  • these bacteria are called merozygotes/partial diploids bc they have 2 copies of a specific gene, resulting in a more complex genetic makeup

lacl-

• defect in the lacl gene

• led to constitutive expression of lac operon, even when lactose was absent

  • basically the continuous expression of the lac operon→ always producing the enzymes needed for lactose metabolism

• these mutations mapped very close to the lac operon

bacterial conjugation

• researchers used bacterial conjugation to introduce different portions of the lac operon onto different strains

• F’ factors (plasmids) were used to transfer parts of the lac operon, such as the lacl gene, into recipient bacteria

merozygotes/partial diploids

• ? instrumental in allowing Jacob, Monod, and Pardee to elucidate the fx of the lacI gene

• 2 key points

  • the 2 lacI genes in a ? may be different alleleles

    • lacI− (mutant allele) on the bacterial chromosome.

    • lacI+ (wild-type allele) on the F’ factor (plasmid).

  • genes on the F’ factor (plasmid) are not physically connected to those on the bacterial chromosome

    • allows the study of how two copies of the lacI gene can interact.

internal activator hypothesis

• Jacob, Monod, and Pardee hypothesized that the lacI- mutation results in the synthesis of an internal inducer

  • internal activator binds to the lacO operator, which activates the transcription of the lac operon, causing constitutive expression

    • e continuous or unregulated expression of a gene or operon

• if correct: the inducer protein produced from the chromosome can diffuse and activate the lac operon on the F’ factor

alternative hypothesis

• the lacI- mutation eliminates the fx of a lac repressor that can diffuse throughout the cell

  • The lac repressor is unable to bind to the lacO operator, allowing RNA polymerase to transcribe the lac operon continuously, regardless of lactose presence

• if correct: the repressor coded on the F’ factor can diffuse and turn off the lac operon on the bacterial chromosome

  • ability of the repressor to diffuse is important bc even when there is a mutated lacI gene on the chromosome (lacI−), the repressor on the F' factor (plasmid) can still function properly to regulate the lac operon and prevent its constitutive expression.

• the correct expression

hypothesis testing

• grow mutant strain and merozygote (partial diploid) strain separately

  • Mutant strain: lacI- (no repressor gene) → Constitutive expression of the lac operon.

  • Merozygote strain: Has two copies of lacI gene, one on the bacterial chromosome (lacI-) and one on the F' plasmid (lacI+), introduced by conjugation.

• divide each strain into 2 tubes

• add lactose to one of the 2 tubes

• incubate the cells long enough to allow lac operon induction

• lyse the cells with a sonicator. This allows beta galactosidase to escape from the cells.

• add β-o-nitrophenylgalactoside (β ONPG)

  • a colorless compound

  • if β-galactosidase is present→ cleave compound to produce galactose and o-nitrophenol (O-NP)→ yellow color

    • O- nitrophenol is yellow, the deeper the yellow color, the more β-galactosidase was produced

    • β-ONPG is a colorless compound that is cleaved by β-galactosidase into galactose and o-nitrophenol (O-NP).

    • If β-galactosidase is present, O-NP will be produced, which is yellow. The intensity of the yellow color will indicate the amount of β-galactosidase activity

• incubate the sonicated cells to allow f β-galactosidase time to cleave β-o-nitrophenylgalactoside

• measure the yellow color produced with a spectrophotometer

  • In the mutant strain (with lacI- mutation), β-galactosidase will be expressed constitutively, meaning yellow color will be produced even without lactose.

  • In the merozygote strain, the expression depends on whether the lacI+ or lacI- is present, with the presence of lactose allowing activation of the lac operon in the lacI+ strain.

the data

• mutant strains express the lac operon at 100% bc of the constitutive expression in the lacl- strain

• merozygote

  • in the absence of lactose, both lac operons are repressed → <1%

  • in the presence of lactose, both lac operons are induced, yielding a higher level of enzyme activity→ 220%

• therefore:

  • lacI- mutant doesn’t make a repressor protein

    • no repressor to bind to the operator (lacO) and block transcription→ continuous production of β-galactosidase, regardless of lactose present/absent

  • lacI+ (WT) gene makes a repressor protein that can repress the lac operon in the same cell.. doesn’t have to be on the same piece of DNA as its gene

    • produce a functional repressor that can regulate the lac operon, even if it's located on a different piece of DNA (e.g., the F' factor/plasmid). The repressor can regulate both operons in a merozygote

trans effect

• interaction btw regulatory proteins and DNA sequences

• genetic regulation that can occur even though DNA segments aren’t physically adjacent

• mediated by genes that code regulatory transcription factors

• ex., action of lac repressor on lac operon

  • lac repressor can bind the operator (lacO) of the lac operon to regulate the lac operon even though it’s located on a different piece of DNA (chromosome or F’ plasmid)

cis affect/cis acting element

• DNA sequence that must be adjacent to the gene(s) it regulates

• mediated by sequences that bind regulatory transcription factors

• ex., the lac operator

  • lac operator (lacO) is DNA sequence near the lac genes (lacZ, lacY, lacA)

  • lacO is a binding site for the lac repressor protein

  • when lac repressor binds lacO, transcription of the lac operon is blocked

  • in presence of lactose or allolactose, the repressor detaches from lacO, allowing the lac operon to be transcribed.

mutation in trans-acting factor vs cis acting element

• mutation in trans acting factor is complemented by the introduction of a second gene with normal function

  • can fix by introducing a second copy of the gene that codes for the normal protein

  • ex., if there is a lacI- mutation (which means the lac repressor is not made), introducing a normal lacI+ gene elsewhere can fix the mutation

• mutation in a cis-acting element is not affected by the introduction of another normal cis-acting element

  • cannot be fixed by introducing another copy of the DNA sequence elsewhere, because the regulation has to happen right next to the gene it controls

  • ex., mutation in the lacO (operator) cannot be rescued by introducing a second lacO somewhere else because the repressor has to bind directly to the lacO that is adjacent to the operon

mutation in trans acting factor

• complemented by the introduction of a second gene with normal function

  • can fix by introducing a second copy of the gene that codes for the normal protein

  • ex., if there is a lacI- mutation (which means the lac repressor is not made), introducing a normal lacI+ gene elsewhere can fix the mutation

mutation in cis-acting element

• not affected by the introduction of another normal cis-acting element

  • cannot be fixed by introducing another copy of the DNA sequence elsewhere, because the regulation has to happen right next to the gene it controls

  • ex., mutation in the lacO (operator) cannot be rescued by introducing a second lacO somewhere else because the repressor has to bind directly to the lacO that is adjacent to the operon

loss of fx mutation in lacI gene or operator site

1. Wild Type:

   • With Lactose: 100% expression (repressor inactivated by lactose).

   • Without Lactose: <1% expression (repressor binds operator).

2. lacI- (mutant):

   • With Lactose: 100% expression (no repressor).

   • Without Lactose: 100% expression (no repressor).

3. lacO- (mutant operator):

   • With Lactose: 100% expression (repressor can't bind).

   • Without Lactose: 100% expression (repressor can't bind).

4. lacI- and lacI+ on F' (merozygote):

   • With Lactose: 200% expression (activator protein boosts transcription).

   • Without Lactose: <1% expression (repressor binds operator).

5. lacO- and lacI- with normal lac operon:

   • With Lactose: 200% expression (activator protein boosts transcription).

   • Without Lactose: 100% expression (repressor can’t bind mutant operator).

Key Points:

• lacI-: Constitutive expression (always on).

• lacO-: No repressor binding, operon always transcribed.

  • Mutant operator (lacO-): The sequence has changed, so the repressor can't bind to it.

  • Result: Since the repressor can't block transcription, RNA polymerase is free to transcribe the lac operon, even in the absence of lactose.

• Activator proteins (CAP) boost transcription when lactose is present.

catabolite repression

• the lac operon can be transcriptionally regulated by an activator protein

• glucose priority

  • when lactose and glucose are both available

    • E coli prefers to use glucose first

    • this prevents the lac operon from being activated and using lactose (? repression)

  • when glucose is depleted, ? repression is relieved, allowing the lac operon to be expressed (so bacteria can use lactose)

• diauxic growth

  • the process where bacteria sequentially use 2 sugars (glucose first, then lactose)

diauxic growth

• the process where bacteria sequentially use 2 sugars (glucose first, then lactose)

diauxic growth experiment

• the process where bacteria sequentially use 2 sugars (glucose first, then lactose)

• steps

  • E coli cells were given glucose and lactose at time zero

  • number of E. coli cells and concentrations of both extracellular glucose and lactose were monitored for 10 hours

• findings

  • cells first used glucose to increase in number

    • glucose used: lac operon inhibited

      • so genes involved in lactose metabolism (lacZ, lacY, lacA) are not transcribed or expressed

      • catabolite repression keeps the lac operon off

  • after glucose was consumed a brief lag phase occurred as cells switched to utilization of lactose

    • lactose used: lac operon is expressed

      • so genes involved in lactose metabolism (lacZ, lacY, lacA) are transcribed and expressed

  • lag phase followed by a second increase in cell number until lactose was eventually depleted and growth leveled off

cyclic AMP cAMP

• a small effector molecule involved in catabolite repression (hint: not glucose)

  • produced from ATP via enzyme adnenylyl cyclase

• binds an activator protein known as the Catabolite Activator Protein CAP

• tldr; low glucose→(E.coli uses ATP via adenylyl cyclase)→ higher cAMP levels

  • cAMP binds CAP→ cAMP-CAP complex→activate lac operon→ allowing transcription of lacZ, lacY, and lacA for lactose metabolization

  • remember; catabolite repression helping glucose preference/priority , (preventing the lac operon from being activated and using lactose) but now you’re low on glucose and need to switch to lactose

cAMP-CAP complex

• an example of transcriptional regulation that is inducible and under positive control

  • binds CAP site near the lac promoter and increases transcription of lacZ, lacY, and lacA for lactose metabolization

• in the presence of glucose, the enzyme adenylyl cyclase is inhibited

  • this decreases cAMP levels in the cell

    • (e.coli uses ATP via adenylyl cyclase to make cAMP)

  • cAMP no longer available to bind CAP

    • transcription rate of genes for lactose metabolism decreases

      • you now have glucose, no need use lactose (you prefer glucose)

lactose, no glucose (high cAMP)

• High transcription: cAMP binds to CAP, enhancing RNA polymerase binding to the promoter, leading to high transcription (lacZ, lacY, and lacA for lactose metabolization).

• Inactive repressor: Allolactose (inducer) binds to the repressor, preventing it from binding to the operator, allowing transcription.

no lactose or glucose (high cAMP)

Low transcription: cAMP is still high, but the repressor remains active and blocks transcription (of lacZ, lacY, and lacA for lactose metabolization) at the operator, even though CAP is present.

• Glucose levels are a key factor in determining cAMP levels. When glucose is absent, adenylyl cyclase (the enzyme that produces cAMP) is not inhibited, leading to high cAMP production

• transcription is low because the lac repressor is still active (since lactose is absent, and thus there's no allolactose to inactivate the repressor). The repressor binds to the operator, blocking RNA polymerase and preventing transcription

lactose and glucose (low cAMP)

Very low transcription: Low cAMP means CAP doesn’t bind effectively, and even though lactose is present, the repressor may still block transcription.

Glucose levels are a key factor in determining cAMP levels. When glucose is present, adenylyl cyclase (the enzyme that produces cAMP) is inhibited, leading to low cAMP production

glucose, no lactose (low cAMP)

• No transcription: Glucose lowers cAMP, so CAP doesn't bind, and the repressor binds the operator, preventing transcription (of lacZ, lacY, and lacA for lactose metabolization)