10.1 The lac operon #
The study of The lac operon provides fundamental principles governing cellular architecture, molecular interactions, and physiological regulation.
Regulation Of Agr Operon
10.1.1 LacI and allolactose #
The lac operon, from Jacob and Monod, is three genes on one mRNA. lacZ encodes Ξ²-galactosidase, which cleaves lactose to glucose and galactose. lacA encodes a transacetylase that is not needed to understand induction. The promoter and the main operator, O1, overlap the start site. Auxiliary operators O2, inside lacZ, and O3, upstream, complete repression. lacI has its own constitutive promoter and is not on the operon mRNA.
The tetramer binds O1 and one auxiliary operator and loops the DNA between them, blocking polymerase. Without lactose, transcription is very low but not zero. A few molecules of permease and enzyme are required for induction, because lactose must enter and the true inducer must be made. That inducer is allolactose, an isomer Ξ²-galactosidase produces as a side reaction, not lactose itself. Allolactose binds LacI and lowers its affinity for the operator. IPTG is a gratuitous inducer: it binds LacI and is not cleaved, so the signal is not consumed.
The genetics separates the protein from the site. In a partial diploid, lacI on one copy represses both: the repressor is trans-acting. An operator mutation Oc that cannot bind LacI drives only the operon on the same DNA: the operator is cis-acting. Is super-repressors ignore inducer and keep the operon off. Promoter mutations are cis-acting and kill transcription even when repressor is absent.
10.1.2 CAP, cAMP, and glucose #
LacI off is not enough for a high rate. The lac promoter matches the consensus poorly, so polymerase binds it badly. CAP, also called CRP, binds as a dimer only when it holds cAMP, at a site centred about 61.5 bp upstream of the start, and contacts the Ξ± subunit to recruit polymerase. The lacUV5 mutation improves the β10 element and largely removes the need for CAP. It does not modify the repressor.
When glucose is in use, cAMP is low, CAP does not bind, and lac transcription stays very low even if lactose is present and LacI has left. When glucose is scarce, cAMP rises and CAP binds, and transcription is high only if LacI is also off.
The link from glucose to cAMP is the phosphotransferase system. During glucose import, Enzyme IIA^Glc is mostly unphosphorylated. That form does not activate adenylate cyclase, and it inhibits LacY, so inducer is excluded. Glucose repression is low CAP activity at the promoter and inducer exclusion at the permease. Both say: do not spend effort on lactose while glucose is available.
| Glucose | Lactose | LacI | CAPβcAMP | lacZYA transcription |
|---|---|---|---|---|
| Present | Absent | Bound | Off | Off |
| Present | Present | Off | Off | Very low |
| Absent | Absent | Bound | On | Off |
| Absent | Present | Off | On | High |
10.2 The trp operon #
The study of The trp operon provides fundamental principles governing cellular architecture, molecular interactions, and physiological regulation.
Trp Operon
10.2.1 Attenuation: where the ribosome sits #
TrpR binds the trp operator only when tryptophan is bound. Tryptophan is a corepressor, so high tryptophan turns repression on, the opposite ligand logic from LacI.
Trp Operon Attenuation
The leader trpL, before trpE, encodes a short peptide with two tryptophan codons in a row. The leader RNA folds in alternative ways. Segments 2 and 3 pair as an antiterminator. Segments 3 and 4 pair as an intrinsic terminator, a GC hairpin plus U. They share segment 3, so only one hairpin exists at a time. Segment 1 can pair with 2 and pause the polymerase so the ribosome can catch up. Coupling is required, because the sensor is that ribosome.
When tryptophan is scarce, Trp-tRNA is scarce and the ribosome stalls on the Trp codons in segment 1. Segment 2 pairs with 3. The antiterminator blocks the terminator, and polymerase continues into the biosynthetic genes. When tryptophan is abundant, the ribosome translates the leader without stalling and covers segment 2. Segment 3 pairs with 4, the terminator forms, and polymerase stops before trpE. If no ribosome translates the leader, the terminator still wins. Attenuation is not “translation stopped, operon on”.
This device needs a ribosome on a nascent transcript, so it does not run on a eukaryotic nuclear mRNA. A riboswitch is the contrast that prevents a mix-up. There the metabolite β thiamine pyrophosphate, FMN, lysine, S-adenosylmethionine, and others β binds the leader RNA directly and shifts terminator against antiterminator, with no ribosome and no repressor protein.
10.3 Positive and negative control #
10.3.1 One contrast #
Negative control means the bound protein turns the promoter off: remove LacI or TrpR, and transcription rises if any positive requirement is also met. Positive control means the bound protein turns the promoter on: remove CAP, and lac stays weak even though LacI has left. An inducer inactivates a repressor, as allolactose does. A corepressor activates a repressor, as tryptophan does. A coactivator is required by an activator, as cAMP is for CAP. AraC does both jobs: it represses the arabinose operon without arabinose and activates it when arabinose is bound. “Positive” and “negative” name the effect of the bound protein, not whether the small molecule is present.
10.4 Key terms, summary, and self-check #
Key terms. LacI; allolactose; O1; CAP; cAMP; inducer exclusion; TrpR; attenuation; antiterminator; corepressor.
Summary. LacI loops O1 to an auxiliary operator and represses lacZYA. Allolactose releases it. CAPβcAMP recruits polymerase to a weak promoter, and only when glucose is scarce. Unphosphorylated Enzyme IIA also blocks LacY. Full expression needs lactose present and glucose absent. At trp, tryptophan is the corepressor for TrpR, while charged tRNA^Trp sets attenuation: a stall on the leader Trp codons allows the 2β3 antiterminator, and a ribosome covering segment 2 allows the 3β4 terminator. Negative control is a bound protein that switches off. Positive control is a bound protein that switches on.
Self-check.
1. Why is allolactose, not lactose, the inducer, and what does it do to LacI?
2. With both glucose and lactose present, where are LacI and CAP, and what second mechanism besides cAMP keeps expression low?
3. In the partial diploid I+ O+ Zβ / Iβ Oc Z+, which lacZ, if either, is constitutive, and why?
4. Which trp leader hairpin forms when the ribosome stalls on the Trp codons, and which forms when Trp-tRNA is plentiful?
5. Why does trp attenuation need coupled translation, while a riboswitch does not?
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