genet 270 lec 15- trp
What are the 7 stages at which gene expression can be regulated in bacteria?
Transcription initiation, elongation, termination, translation initiation, elongation, termination, and protein activity (stability, location, modification).
What is the single most common level of regulation in bacteria?
Transcription initiation, because it prevents waste and energy loss.
What are 4 differences between catabolic vs anabolic (biosynthetic) operons?
Catabolic: induced by substrate, effector = inducer, repressor loses DNA binding when inducer present.
Anabolic: repressed by product, effector = corepressor, repressor gains DNA binding when corepressor present.
What 2 states can repressors of biosynthetic operons exist in?
Aporepressor (no effector bound, cannot repress) and corepressor-bound repressor (active DNA-binding form).
What are the 5 structural genes of the trp operon and what do they encode collectively?
trpE, trpD, trpC, trpB, trpA encode enzymes of the tryptophan synthesis pathway.
Where is trpR located relative to the trp operon and what does it encode?
trpR is unlinked (separate part of the genome) and encodes the TrpR repressor protein.
What is the effector molecule for the trp operon’s repressor and what type of control does this represent?
Tryptophan, acting as a corepressor in a negative repression system.
What are the 3 mechanistic steps for repression of trp operon transcription initiation?
Tryptophan binds TrpR → induces active conformation
TrpR–tryptophan complex binds trpO (overlaps promoter)
Binding physically blocks RNA polymerase from initiating transcription.
What happens in the absence of tryptophan at the operator? (2 outcomes)
TrpR remains an aporepressor and cannot bind DNA, so the operon is ON and full-length mRNA is produced.
What are the 2 mutations that cause constitutive trp operon expression?
trpR⁻ (defective repressor) and trpOᶜ (operator mutation preventing binding).
What are the 3 defining features of attenuation?
Transcription begins normally, may be terminated in the leader region, and is controlled by translation-dependent changes in mRNA secondary structure.
What 3 pieces of genetic evidence support attenuation?
trpR⁻ mutants still show partial regulation
Mutations lowering tRNAᵗʳᵖ increase transcription
Deleting the leader region abolishes regulation.
What are the 4 mRNA regions (1–4) in the trp leader and what can they form?
Regions 1, 2, 3, 4 form alternative hairpins: 1:2, 2:3, and 3:4 (terminator).
What is unique about the trp leader peptide and how does this relate to regulation? (2 features)
It encodes a short peptide with two adjacent Trp codons; ribosome stalling here signals low tryptophan, altering hairpin formation.
What are the 4 mechanistic events leading to attenuation at high tryptophan levels?
tRNAᵗʳᵖ abundant → ribosome quickly translates to stop codon
Ribosome blocks region 2
Regions 3 & 4 pair → terminator hairpin forms
Transcription stops early (~140 nt).
What are the 5 mechanistic events at low tryptophan levels?
tRNAᵗʳᵖ scarce → ribosome stalls at Trp codons
Region 1 blocked but region 2 free
Regions 2 & 3 pair → anti-terminator
Region 4 cannot pair with 3
Transcription continues through structural genes.
What 3 rules determine which mRNA hairpin forms?
Ribosome position on the leader
Region availability
Relative timing of transcription vs translation.
What 2 mutations cause constitutive termination in the leader region?
Mutations that prevent 2:3 pairing (favoring 3:4) or mutation of AUG start codon, preventing ribosome loading → persistent 1:2 → terminator forms
What observation shows attenuation is a general mechanism, not trp-specific?
Starvation for other amino acids (e.g., Arg) also causes ribosome stalling in corresponding leader peptides → attenuation phenotypes.
What 3 combined systems regulate the trp operon?
Repression at initiation, attenuation, and amino acid–dependent translation rates.
A trpR⁻ mutant is grown in high tryptophan. What happens to transcription?
Repression fails, but attenuation still works, so transcription decreases but is not fully OFF.
A mutation removes trpO. What phenotype do you expect?
Constitutive high expression because repression at initiation no longer occurs.
Region 3 of the leader is deleted. What happens?
Neither 3:4 (terminator) nor 2:3 (anti-terminator) can form → attenuation control collapses → transcription becomes unregulated.
A ribosome stalls at the Trp codons. What does RNAP do next?
It continues transcription because 2:3 anti-terminator forms, preventing 3:4 terminator
tRNAᵗʳᵖ is abundant but tryptophan is low. What will happen?
Ribosome quickly reads Trp codons → 3:4 terminator forms → attenuation despite low Trp.
Mutate the leader start codon (AUG→AUA). Predict expression.
Ribosome never loads → region 1:2 hairpin stable → constitutive termination (always OFF).
Region 2 is deleted. What forms: terminator or anti-terminator?
Region 2 cannot pair with 3 → 3:4 always forms → operon always attenuated.
RNAP is slowed down artificially. What happens?
Ribosome catches up → activation of attenuation (more terminator formation).
Ribosome is slowed (e.g., another AA starvation). Effect?
Ribosome stalls early → 2:3 pairing → transcription continues through operon. (General attenuation mechanism.)
Add tryptophan suddenly during transcription. What happens?
Ribosome quickly finishes leader peptide → blocks 2 → terminator hairpin forms → transcription stops early.
CRISPR deletes region 4. Predict the output.
No terminator can form → attenuation abolished → operon always ON unless repressed at initiation.
A mutation increases TrpR affinity for DNA even without tryptophan.
Operon becomes non-inducible, similar to lacIˢ in the lac operon.
A deletion removes the entire leader region. What happens?
Attenuation disappears → operon controlled only by repression at initiation
A strain has normal trpR but defective tryptophanyl-tRNA synthetase. Predict expression.
tRNAᵗʳᵖ remains uncharged → ribosomes stall → no attenuation, operon strongly ON.
Mutate region 1 so it cannot pair with region 2.
Region 2 always pairs with 3 → anti-terminator always forms → operon constitutively ON.