ch 11 (lectures 21-23) Flashcards
Why do cells regulate gene expression, and how is it achieved?
Cells regulate gene expression to:
* Produce different protein combinations for different activities
* Conserve energy by shutting off unused genes
* Recognize environmental conditions
* Respond by turning specific genes on or off.
This is achieved through regulatory proteins that control transcription.
How do regulatory proteins control transcription?
Regulatory proteins control transcription by influencing RNA polymerase (RNA pol) activity. For RNA pol to transcribe, it must bind to a promoter (recruitment) and move downstream without dissociating.
What roles do promoters, repressors, activators, and operators play?
Repressors block transcription by preventing RNA pol recruitment or blocking its movement.
Activators promote transcription by helping recruit RNA pol and stabilizing it at the promoter.
Operators are DNA sequences in prokaryotes that bind repressors.
Activator binding sites are specific DNA sequences where activators bind.
What are allosteric effectors, and how do they influence transcription regulation?
Allosteric effectors are small molecules that bind to regulatory proteins at a specific site called the allosteric site, causing a conformational (structural) change. This change can either allow or prevent the protein (activator or repressor) from binding to DNA.
* Since activators and repressors must bind DNA to function, preventing this binding blocks their activity. When an effector positively regulates the protein’s activity, it’s called an inducer. Inducers can act in different ways depending on the specific protein and effector—either promoting or blocking transcription.
What is an operon, and how does it allow bacteria to produce multiple proteins?
An operon is a group of genes regulated together in bacteria- they are controlled by the same promoter and transcribed into one mRNA molecule.
* Genes in an operon are coordinately controlled (turned on/off together).
Ribosomes can start translation at internal sites on the mRNA.
* This allows production of more than one protein from a single mRNA.
Common in prokaryotes like bacteria.
What’s the difference between an operon and a single gene in terms of protein synthesis?
Single Gene:
* One gene = one open reading frame (ORF)
* mRNA has only one ribosome binding site
* Ribosome initiates at a single site
* Produces only one protein
Operon:
* Multiple genes grouped together under one promoter
* Transcribed as a single mRNA
* Ribosomes can initiate at internal sites on the mRNA
* Produces more than one protein (polygenic)
What is the lac operon, and how is it controlled by a repressor protein?
The lac operon is required for lactose metabolism in bacteria.
It contains genes that:
* Transport lactose into the cell
* Break down lactose for energy
A repressor protein controls the lac operon by:
* Binding the operator when lactose is absent
* Blocking transcription of lac genes
When lactose is present, it acts as an inducer, binding the repressor and inactivating it, allowing transcription.
Why is the lac operon turned off when lactose is not present?
β-galactosidase (β-gal) breaks down lactose into two sugars.
The lac operon produces enzymes that:
* Import lactose into the cell
* Break it down
If no lactose is present, making these enzymes is wasteful.
* Therefore, the cell turns off the lac operon to save energy.
How does β-galactosidase (β-gal) and allolactose regulate the lac operon?
β-galactosidase (β-gal):
* Breaks down lactose into glucose and galactose
* Also converts some lactose into allolactose
Allolactose:
* Acts as an allosteric effector and inducer
* Binds to the repressor, causing it to release from the operator
* This activates the lac operon, allowing transcription
Result:
In the presence of lactose, allolactose forms → lac operon is turned on
How does IPTG compare to lactose in regulating the lac operon?
Lactose:
Natural inducer of the lac operon
Converted to allolactose by β-galactosidase
Eventually broken down by β-gal
IPTG:
Synthetic inducer
Mimics allolactose by binding to the lac repressor (I)
Not broken down by β-galactosidase → provides sustained induction
What do constitutive mutants and cis/trans acting elements tell us about lac operon regulation?
Constitutive mutant:
* The operon is always on, even without an inducer
* Example: mutation in the operator that prevents repressor binding
Cis-acting elements:
* Affect gene expression only on the same DNA molecule
* Typically refer to DNA sequences, like promoters or operators
Trans-acting factors:
* Can affect other DNA molecules
* Usually proteins (like the lac repressor) that can diffuse throughout the cell
Example case:
* Z gene (β-galactosidase) is constitutively expressed
* Y gene (permease) is not, indicating operator mutation affects Z in cis
What promoter elements are important for RNA polymerase binding in prokaryotes, and how was this discovered?
RNA polymerase binds to specific promoter sequences
Two key regions:
* –10 element
* –35 element
Mutational analysis of the lac operon showed these regions are critical for transcription initiation in prokaryotes
Mutations in these elements reduce or prevent transcription
What is the operator in the lac operon, and what did Oc mutations reveal about its function?
The operator is a specific DNA sequence that binds the lac repressor protein
Oc mutations (operator-constitutive) cluster just downstream of the lac promoter
* These mutations prevent repressor binding → operon is always on
The operator region was shown to be partially transcribed
IPTG causes the repressor to unbind from the operator
The operator includes palindromic sequences, allowing repressor dimer binding
How do glucose levels regulate the lac operon in E. coli?
Glucose is the preferred energy source (easier to metabolize than lactose)
If glucose is present, the cell shuts off the lac operon, even if lactose is also present
* This type of regulation is called catabolite repression
Key players:
* Adenylate cyclase: converts ATP to cAMP
* Low glucose → high cAMP
* High glucose → low cAMP
cAMP binds to CAP (catabolite activator protein)
* cAMP–CAP complex helps activate the lac operon
Without cAMP (high glucose), CAP cannot bind → lac operon is off
How does CAP binding help RNA polymerase activate the lac operon?
CAP binds DNA upstream of the lac promoter
* Binding causes the DNA to bend
* This bending helps RNA polymerase bind to the promoter
CAP also directly contacts RNA polymerase, stabilizing the complex
Together, these actions enhance transcription of the lac operon when glucose is low
How is the trp operon regulated when tryptophan is abundant?
Tryptophan acts as an allosteric effector
* Binds the Trp repressor protein
* Causes a conformational change, allowing it to bind the operator
* This blocks transcription of the trp operon
Attenuation also helps reduce transcription:
* High Trp → ribosome moves quickly → region 3 pairs with 4
* This forms a terminator hairpin → transcription stops
What is attenuation in the trp operon and how does it work?
A secondary regulatory mechanism based on mRNA structure
The leader sequence has 4 regions (1–4) that can base-pair
The ribosome’s speed determines which regions pair:
* High Trp → fast translation → region 3 pairs with 4 → terminates transcription
* Low Trp → slow/stalled translation → region 2 pairs with 3 → transcription continues
Why is attenuation only possible in bacteria?
Because in prokaryotes, transcription and translation are coupled
* Ribosomes start translating mRNA while it’s still being transcribed
* This coupling allows the ribosome to influence RNA folding in real-time
What determines whether bacteriophage λ enters the lytic or lysogenic cycle?
Phage λ is a temperate phage → can follow two paths:
Lytic cycle → phage replicates, host cell is lysed
Lysogenic cycle → phage DNA integrates into host genome as a prophage
Decision depends on host environment:
Abundant resources → favors lytic cycle (phage replication + cell lysis)
Limited resources → favors lysogenic cycle (phage DNA stays dormant)
What do cloudy vs. clear bacteriophage plaques indicate?
Plaques form when phages infect and lyse bacterial cells growing on a plate.
The appearance of plaques tells us about the phage’s life cycle:
* Cloudy plaques → some bacteria survive
* Indicates wild-type temperate phage
Some phages enter the lysogenic cycle, sparing host cells
* Clear plaques → all bacteria lysed
* Indicates mutant phage that can only undergo the lytic cycle
* Every infected cell is destroyed, leaving a completely clear zone
How is gene expression organized in the phage lambda (λ) genome, and when is the lytic vs. lysogenic decision made?
Phage λ has a ~50 kb genome, with genes expressed in a logical sequence
Gene expression begins at two key promoters:
* PR (rightward promoter)
* PL (leftward promoter, not mentioned here but typically paired with PR)
The decision between lytic and lysogenic occurs early after infection
* Controlled by transcription from PR (lytic) and PRM (promoter for repressor maintenance, lysogenic)
The regulatory proteins expressed from these promoters determine the path:
* High Cro expression → lytic cycle
* High CI repressor expression → lysogenic cycle
How does the battle between the PR and PRM promoters determine the phage λ life cycle?
The decision point occurs at two opposing promoters:
PR → promotes expression of** cro**, leading to the lytic cycle
PRM → promotes expression of λ repressor (CI), leading to lysogeny
Cro protein:
* Inhibits PRM → shuts down CI expression
* Favors lytic gene expression
CI (λ repressor) protein:
* Inhibits PR → blocks cro expression
* Maintains lysogenic state
Whichever protein dominates early determines the outcome:
* Cro wins → Lytic cycle
* CI wins → Lysogenic cycle
How do λ repressor and Cro proteins interact with the operator sites OR1–OR3 to control the lytic vs. lysogenic decision?
The operator region has three sites: OR1, OR2, OR3
These sites overlap promoters:
* OR1 overlaps PR (lytic promoter)
* OR3 overlaps PRM (lysogenic promoter)
Binding affinities differ for each protein:
λ repressor (CI): OR1 > OR2 > OR3
* Binds OR1 first → blocks PR (prevents cro expression)
* Also activates PRM (enhances own expression) via OR2
* Lysogeny is reinforced
Cro: OR3 > OR2 > OR1
* Binds OR3 first → blocks PRM (prevents CI expression)
* Lytic genes at PR are transcribed
* Promotes the lytic cycle
How do nutrient levels in the host cell influence the battle between PRM and PR in phage λ?
Low nutrients (starvation) → favors lysogeny
* λ repressor is stable and accumulates
* Binds OR1 first, blocking PR (shuts off cro/lytic genes)
* PRM is free to recruit RNA pol → more λ repressor (CI) made
* No Cro → Lysogenic state maintained
High nutrients → favors lytic growth
* λ repressor is unstable → levels are low
* Cro binds OR3 first, blocking PRM (shuts off cI/λ repressor)
* PR recruits RNA pol → more Cro → virulence genes expressed
* No CI → Lytic cycle proceeds
Initial state:
* Both Cro and λ repressor are expressed briefly after infection
* Metabolic state of the cell determines which protein dominates via stability
