Unit 26 Question Bank
It yields POLYCISTRONIC mRNA — transcribed into one large mRNA molecule containing multiple independent translation start (AUG) and stop (UAA) codons for each cistron — and therefore gives coordinate expression of all its genes. A cistron is the genetic unit encoding one polypeptide: the “one cistron, one subunit” concept, refining “one gene, one enzyme”.
The lac operon is the model: lacZ (β-galactosidase), lacY (a permease, for the permeation of lactose into the cell) and lacA (a thiogalactoside transacetylase), with lacI, encoding the repressor, outside the operon.
Operons do not occur in eukaryotes, because cap-dependent scanning permits only one translation initiation site per mRNA.TMU Lecture 24 · Harper's ch.38
Structural genes: lacZ — β-galactosidase; lacY — a permease, the permeation of lactose into the cell; lacA — a thiogalactoside transacetylase, transcribed as one polycistronic mRNA.
Negative control: lacI encodes the lac operon repressor protein — four identical subunits of molecular weight 38,000, with high affinity for the operator locus. The operator is a region of double-stranded DNA 27 base pairs long with twofold rotational symmetry — an inverted palindrome; LacI binding covers 17 base pairs, and only two subunits appear to bind. Addition of lactose or of a gratuitous inducer such as IPTG causes derepression.
Positive control: catabolite gene activator protein (CAP), also called cAMP regulatory protein (CRP), in conjunction with cAMP.
Maximal activity requires lactose present AND glucose absent: the operon has one regulator that acts positively (the cAMP-CRP complex) and one that acts negatively (the LacI repressor). Induction is fast — mRNA fully induced within 5–6 minutes, β-galactosidase maximal within 10.TMU Lecture 24 · Harper's ch.38
When bacteria are exposed to both lactose and glucose as sources of carbon, they first metabolize the glucose and then temporarily stop growing, until the genes of the lac operon become induced and they gain the ability to metabolize lactose as a usable energy source.
Mechanism: it is mediated by a catabolite gene activator protein (CAP) in conjunction with cAMP. When glucose or glycerol is present in concentrations sufficient for growth, the bacteria lack sufficient cAMP to bind to CAP, because the glucose inhibits adenylyl cyclase, the enzyme that converts ATP to cAMP. Without cAMP, CAP cannot bind DNA and cannot activate transcription.
The CAP-cAMP regulator acts as a POSITIVE regulator, so the operon is under dual control — an AND gate requiring lactose present and glucose absent.TMU Lecture 24 · Harper's ch.38
Positive regulation: the regulatory element is present and expression is increased; that mediating positive regulation is a positive regulator or ACTIVATOR.
Derepression is removal of a repressor, and works because a double negative has the effect of acting as a positive.
Related terms: inducible genes have relatively low basal rates of transcription; housekeeping genes are expressed constantly; a constitutive mutation makes an inducible gene expressed permanently.
The lac operon uses both logics at once — one regulator acting positively (cAMP-CRP) and one acting negatively (LacI).TMU Lecture 24 · Harper's ch.38
Histone acetylation and deacetylation occur on lysine residues in the amino terminal tails of histone molecules. Acetylation reduces the positive charge of these tails, decreases the binding affinity of histone for the negatively charged DNA, and causes disruption of nucleosomal structure, allowing access of transcription factors to cognate regulatory DNA elements and enhancing binding of the basal transcription machinery to the promoter. Histone deacetylation has the opposite effect.
Methylation of deoxycytidine residues in the sequence 5′-mCpG-3′ silences genes: in mouse liver, only the unmethylated ribosomal genes can be expressed, and many animal viruses are not transcribed when their DNA is methylated.
Because both marks are copied through cell division, this level of control is heritable — a liver cell's daughters are liver cells.TMU Lecture 24 · Harper's ch.38
General principles: binding must be of high affinity to the specific site and of low affinity to other DNA; binding is usually by a dimer, matching the twofold symmetry of a palindromic site; and the protein-DNA interactions are maintained by hydrogen bonds and van der Waals forces.
Helix-turn-helix — illustrated by Cro: three antiparallel β sheets and three α-helices, with α3 and α2 held at about 90 degrees to each other by a turn of four amino acids; the α3 helix is the DNA recognition surface. In the dimer, the two recognition helices are 34 Å apart — one turn of B-DNA.
Zinc finger — a domain folded around a zinc ion that stabilises a short recognition helix.
Leucine zipper — a helical wheel of seven spokes corresponding to the seven amino acids, with leucine at every seventh position; the zipper mediates dimerisation while an adjacent basic region binds DNA.
All three converge on the same solution: presenting an α-helix to the major groove.TMU Lecture 24 · Harper's ch.38
Why it matters
Analysis of lactose metabolism in E. coli led to the operon model, and prokaryotes provide models for the study of gene expression in mammalian cells. The vocabulary of this one system — promoter, operator, repressor, activator, induction, cis and trans — is the vocabulary of the whole field.
Structure
An operon can be regulated by a single promoter or regulatory region. The lac operon comprises three structural genes:
- lacZ — β-galactosidase, which cleaves lactose
- lacY — a permease, for the permeation of lactose into the cell
- lacA — a thiogalactoside transacetylase
These are transcribed into one large mRNA molecule containing multiple independent translation start (AUG) and stop (UAA) codons for each cistron — a polycistronic mRNA giving coordinate expression. Translation can occur even before transcription is completed.
lacI lies outside the operon with its own promoter and encodes the lac operon repressor protein.
Negative control — the repressor
The repressor has four identical subunits of molecular weight 38,000 and a high affinity for the operator locus. The operator is a region of double-stranded DNA 27 base pairs long, with a twofold rotational symmetry and an inverted palindrome; LacI repressor binding covers 17 base pairs, and only two subunits of the repressor appear to bind.
The palindrome is not incidental. A protein with twofold rotational symmetry must bind a DNA site with twofold rotational symmetry — a rule that recurs at the λ operator and at every dimeric transcription factor in this unit.
Addition of lactose or of a gratuitous inducer such as IPTG causes the inducer to bind the repressor, which releases the operator: an inducer derepresses the lac operon. Because this is a double negative acting as a positive, the gene is off by default and needs no continuous input to stay off. Induction is fast — lac-specific mRNAs are fully induced within 5–6 minutes, and β-galactosidase protein is maximal within 10 minutes (a type A response).
Positive control — catabolite repression
When bacteria are exposed to both lactose and glucose as sources of carbon, they first metabolize the glucose and then temporarily stop growing, until the genes of the lac operon become induced. This is catabolite repression, mediated by a catabolite gene activator protein (CAP) in conjunction with cAMP — also referred to as the cAMP regulatory protein (CRP).
When glucose or glycerol is present in concentrations sufficient for growth, the bacteria will lack sufficient cAMP to bind to CAP, because the glucose inhibits adenylyl cyclase, the enzyme that converts ATP to cAMP. The CAP-cAMP regulator acts as a positive regulator.
The two signals together
| Lactose | Glucose | Repressor | CAP-cAMP | Transcription |
|---|---|---|---|---|
| Absent | Present | Bound | Inactive | Off |
| Absent | Absent | Bound | Active | Off |
| Present | Present | Released | Inactive | Very low |
| Present | Absent | Released | Active | MAXIMAL |
The logic is worth stating explicitly: the repressor asks “is there lactose to eat?” and CAP-cAMP asks “is there anything better?” The operon commits only when the answer is lactose yes, glucose no. It is an AND gate assembled from one negative and one positive regulator — one that acts positively (the cAMP-CRP complex) and one that acts negatively (the LacI repressor).
Mutations — and the cis/trans distinction
- lacI mutated so LacI cannot bind operator DNA → constitutive expression.
- lacI mutated so LacI cannot bind the inducer → the operon can never be induced — permanently repressed.
- Operator mutated so it cannot bind a normal repressor → constitutive expression, but acting only in cis. lacI encodes a diffusible protein and acts in trans, so a normal copy elsewhere rescues it; an operator is a DNA site and cannot be rescued.
The organising difference
A bacterium regulates to adapt; a eukaryote regulates to differentiate. A bacterium must respond to a sugar appearing in its medium within 5–6 minutes and reverse the decision as quickly; a liver cell must keep the muscle genes off permanently, through every division, for the life of the organism. Almost every difference below follows from that.
| Prokaryote | Eukaryote | |
|---|---|---|
| Gene organisation | Operons — polycistronic mRNA, coordinate expression | One gene, one mRNA |
| Chromatin | None | Nucleosomes — an additional level of control |
| DNA methylation | Restriction/modification | 5′-mCpG-3′ silences genes, heritably |
| Distance of control | Operator adjacent to promoter | Enhancers at 1000–10⁵ bp, either orientation |
| Transcription/translation | Coupled | Separated by the nuclear membrane |
| Post-transcriptional control | Limited | Splicing, alternative promoters, RNA editing, miRNA, stability |
Why eukaryotes cannot use operons
Polycistronic mRNA requires a ribosome to initiate internally. Eukaryotic ribosomes cannot: the 5′ cap directs the 40S subunit to the 5′ end, which then scans to the first suitable AUG. One entry point means one protein per message, so related genes must be coordinated by shared regulatory elements rather than by physical clustering.
The distinctively eukaryotic mechanism — chromatin
Chromatin remodeling is an important aspect of eukaryotic gene expression; chromatin structure provides an additional level of control of gene transcription, and it is what produces differential expression between tissues.
- Histone acetylation and deacetylation occur on lysine residues in the amino terminal tails of histone molecules. Acetylation reduces the positive charge of these tails, decreases the binding affinity of histone for the negatively charged DNA, and disrupts nucleosomal structure, giving access of transcription factors to cognate regulatory DNA elements and enhancing binding of the basal transcription machinery to the promoter. Deacetylation has the opposite effect.
- Methylation of deoxycytidine residues in the sequence 5′-mCpG-3′ silences genes: in mouse liver, only the unmethylated ribosomal genes can be expressed, and many animal viruses are not transcribed when their DNA is methylated.
The decisive property is that both marks are copied through cell division. A repressor protein must be continuously present and is diluted at every division; packaging a gene away makes it off by default, at no ongoing cost, and heritably.
Enhancers and combinatorial control
Certain DNA elements enhance or repress transcription of eukaryotic genes, acting at up to 10⁵ bp and in either orientation, the intervening DNA looping out. The β-interferon gene, induced upon viral infection, has an enhancer between nucleotides −110 and −45 containing four distinct clustered cis elements, bound by NF-κB, an IRF family member, the heterodimeric leucine zipper factor ATF-2/c-Jun, and the ubiquitous architectural factor HMG I(Y). HMG I(Y) induces a significant bend in the DNA, and along with the three trans factors, by inducing a series of critically spaced DNA bends, brings about the formation of a unique, stereospecific, three-dimensional structure in which all four factors are active — the enhanceosome, which fires only when all four signals coincide.
Combinations of DNA elements and associated proteins provide diversity in responses, and tissue-specific expression may result from the action of enhancers or repressors.
What is shared
The underlying logic is identical. Both use positive and negative regulation through the interaction of specific binding regulatory proteins with various regions of DNA; both distinguish cis elements from trans factors; and both bind DNA through the same helix-turn-helix, zinc finger and leucine zipper motifs. The eukaryotic cell did not replace the bacterial logic — it added layers on top of it.