genet 270 lec 14- lac
What are the 3 main reasons bacteria regulate gene expression?
To adapt to environmental changes, maximize growth efficiency, and avoid wasting energy by producing proteins only when needed.
What are the 2 levels at which bacteria can regulate gene expression?
They regulate by controlling RNA levels and by on/off regulation (induction or repression) of genes or operons.
What are the 3 major levels of regulation in bacterial gene expression?
Transcriptional, translational, and post-translational regulation, each affecting different stages of gene expression.
What is the single most common regulatory point in bacteria? (1 concept)
Transcription initiation, because it is the most energy-efficient point of control.
What 7 molecular mechanisms can regulate bacterial gene expression?
Alternative sigma factors, repressors, activators, antisense RNAs, RNA secondary structure, RNA stability, and protein stability/modification.
What are the 3 defining features of a repressor protein?
It binds the operator, often overlaps the promoter, and turns off transcription (negative regulation).
What are the 3 defining features of an activator protein?
It binds an activator site upstream of −35, helps recruit RNA polymerase, and turns on transcription (positive regulation).
What are the opposite mutant effects seen in positive vs negative regulation? (2 comparisons)
In negative regulation, loss-of-function is constitutive and recessive; in positive regulation, loss-of-function is non-inducible and recessive.
What are the 3 effects of a gain-of-function mutation in a repressor vs activator?
Repressor GOF → non-inducible; Activator GOF → constitutive; both behave dominantly
What were the 2 experimental tools used to isolate lac mutants?
MacConkey lactose plates (white = Lac⁻, pink = Lac⁺) and X-gal plates (blue for β-gal activity).
What were the 2 steps used to create merodiploids?
They transferred mutations to an F′ lac plasmid via recombination and formed partial diploids by conjugation.
What 3 questions does complementation analysis answer?
Number of gene products, dominant vs recessive, and cis vs trans nature of mutations.
What are the 2 structural genes of the lac operon and their functions?
lacZ (β-galactosidase) and lacY (lactose permease).
What 2 observations showed lac expression is inducible?
Low lacZ/Y expression without lactose and high expression when lactose (inducer) is present.
What are the 2 classes of constitutive lac mutants?
lacI⁻ (repressor null) and lacOᶜ (operator constitutive).
What are the 3 defining features of lacI⁻ mutants?
They are constitutive, recessive, and complemented by lacI⁺ because LacI acts in trans.
What 3 features define lacOᶜ mutants?
They are dominant, cis-acting, and cannot be complemented because the operator is DNA, not a protein.
What are the 4 key components of the lac operon model (classic view)?
Structural genes Z/Y/A, promoter lacP, operator lacO, and repressor LacI.
What are the 3 molecular events that occur when lactose is present?
Lactose → allolactose, allolactose binds LacI → LacI dissociates → RNAP transcribes lacZ/Y/A.
What 3 mutant types produce a non-inducible phenotype?
lacZ⁻/lacY⁻, lacIˢ (super-repressor), and lacP⁻ (promoter mutant).
What are the 2 functional defects in LacIˢ mutants?
They fail to bind inducer or bind it but cannot release from the operator → always repress. Dominant
What are the 3 modern refinements of the lac operon?
Third gene lacA, true inducer allolactose, and LacI binding to three operators (O₁, O₂, O₃) enabling DNA looping.
What 2 mutant classes revealed positive regulation of the lac operon?
crp⁻ and cya⁻, which produce Lac⁻ and non-inducible phenotypes.
What are the 3 molecular components of positive regulation?
CRP (activator protein), cAMP (effector), and the CRP-binding site upstream of Plac.
What 4 conditions must be met for maximum lac operon expression?
Lactose present, LacI inactivated, low glucose, high cAMP → CRP bound.
What are the 2 reasons CRP–cAMP acts as a global regulator?
It activates many sugar operons, and ensures preferred carbon sources (glucose) are used before lactose.
A lacI⁻ mutant is grown with and without lactose. What phenotype do you expect and why?
Constitutive expression because lacI⁻ makes no functional repressor, so the operator is always free and lacZ/Y are always transcribed. LacI acts in trans, so only the mutant chromosome is affected.
A lacI⁻ mutation is complemented by an F’ lacI⁺ plasmid. What is the phenotype and what does this show?
The merodiploid becomes inducible, showing that LacI protein is diffusible (trans-acting) and can repress both chromosomal and plasmid operons
A lacOᶜ mutant is complemented with a wild-type operator on an F’ plasmid. Does the lacOᶜ phenotype disappear?
No — lacOᶜ remains constitutive because the operator is a cis-acting DNA site, not a protein. Only the operon on the mutant DNA is permanently ON.
In a merodiploid F’ lacOᶜ lacZ⁻ / lacO⁺ lacZ⁺, will β-galactosidase be inducible or constitutive?
Inducible, because the only functional lacZ⁺ gene sits next to the normal operator (lacO⁺). The lacOᶜ chromosome has lacZ⁻, so even though it is constitutive, no β-gal is produced.
A lacIˢ (super-repressor) mutant is present with lactose. What happens to lac expression and why?
Operon remains non-inducible because LacIˢ repressor cannot bind inducer (allolactose), so it never dissociates from the operator. Dominant over lacI⁺.
A lacP⁻ promoter mutant is present. What is the phenotype even if lactose is added?
Non-inducible, because RNA polymerase cannot bind the mutated promoter, so transcription cannot occur regardless of repressor state.
A strain is lacI⁻ and lacO⁺. What phenotype results and what type of regulation does this illustrate?
Constitutive expression, demonstrating negative regulation, where loss of a repressor = operon always ON (page 9 diagram).
A strain is lacI⁺ but carries lacOᶜ. What is the phenotype and what does this reveal about regulatory sites?
constitutive, because lacOᶜ prevents repressor binding regardless of whether repressor is present. Shows the operator is a cis-dominant DNA element.
A crp⁻ mutant is given lactose. Will the lac operon be induced? Why or why not?
No, because CRP is required for positive regulation via CRP–cAMP activation. Without CRP, RNA polymerase cannot efficiently initiate transcription from Plac.
A cya⁻ mutant (no adenylate cyclase) is grown in lactose but low glucose. What happens to lac expression?
Non-inducible, because cya⁻ cannot make cAMP, so CRP never activates. Repressor may be inactivated by lactose, but transcription remains too inefficient to turn ON.
Two sugars are present: lactose and glucose. Predict lac operon activity.
Mostly OFF, because glucose keeps cAMP levels low → CRP is inactive, so positive regulation cannot occur even though lactose removes LacI. This is catabolite repression.
Lactose is present but glucose is absent. What two molecular events activate lac transcription?
(1) Allolactose binds LacI → LacI dissociates from operator.
(2) cAMP rises → binds CRP → CRP activates RNAP at Plac. Strong induction only occurs when both conditions are met.
Predict operon activity in −lactose / −glucose conditions.
OFF because lactose is absent → LacI remains bound. Even though cAMP is high and CRP active, negative regulation overrides positive regulation.
A strain has perfect LacI and CRP systems but a mutation deletes the CRP binding site upstream of Plac. What is the outcome?
Low-level, weak induction, because the operon can only rely on repressor inactivation; CRP cannot recruit RNA polymerase without its binding site.
A strain contains lacOᶜ, lacIˢ, and lactose is present. What happens?
Constitutive expression, because lacOᶜ prevents any repressor (even LacIˢ) from binding. The cis-dominant operator mutation overrides trans-acting changes.
If lacZ⁻ and lacY⁻ mutations occur, but lacA is functional, what phenotype results on lactose medium?
Lac⁻, because lacZ and lacY are both required for lactose transport (Y) and cleavage (Z); lacA alone cannot enable lactose metabolism.