Control of gene expression

One genome, two hundred cell types

A human body runs on something like twenty thousand protein-coding genes and contains over two hundred recognisably different kinds of cell. Every one of those cells, apart from mature red blood cells and the gametes, carries the whole genome, and transcribes perhaps half of it at any moment. Which subset is transcribed accounts for most of the differences between cell types.

Gene expression can be controlled at every stage between a gene and its protein: transcription blocked or accelerated, pre-mRNA spliced one way rather than another, mRNA destroyed before a ribosome reaches it, the finished polypeptide left inactive until it is wanted. Most regulation, and most of the regulation on these specifications, acts at transcription, because a process not started consumes no substrates.

Gene expression

The process by which the information in a gene is used to produce a functional product, usually a polypeptide.

Transcription factor

A protein that binds to a specific base sequence in DNA, usually in or near a promoter region, and so increases or decreases the rate of transcription of that gene.

Promoter

A region of DNA at the start of a gene to which RNA polymerase and transcription factors bind.

A transcription factor is a protein, so it is the product of a gene and is itself regulated by other transcription factors. Regulation in a eukaryote is therefore layered, and one signal arriving at the cell surface can change the transcription of hundreds of genes.

Differentiated cells retain the whole genome, and two experiments demonstrate it. A nucleus taken from an adult mammary gland cell and transferred into an egg cell whose own nucleus had been removed produced a whole sheep, born in 1996. Induced pluripotent stem cells, reported in 2006, were produced from differentiated cells using four transcription factors.

Transcription factors: the general mechanism

The mechanism is the same in each case. A transcription factor has a region whose tertiary structure is complementary to a specific short sequence of bases, usually in or beside the promoter of the gene it controls. It binds there by hydrogen bonds and ionic interactions with the exposed edges of the bases. Once bound, it either helps RNA polymerase attach and start transcribing, or gets in the way so that RNA polymerase cannot. Factors that increase the rate are activators and those that decrease it are repressors; both are transcription factors, so define a transcription factor as altering the rate of transcription rather than as switching genes on.

The specificity is of the same kind as an enzyme's: a binding site with a particular shape and charge distribution fits one short stretch of DNA in three billion base pairs closely enough for binding to persist. A change in the factor's shape removes that complementarity or creates it. Each example below applies that mechanism.

The lac operon

Escherichia coli uses glucose preferentially but can metabolise lactose, using enzymes that transport lactose into the cell and hydrolyse it. Synthesising those enzymes in the absence of lactose would consume amino acids and ATP without benefit, so the genes are transcribed at a low rate until lactose is present. The control circuit was described by François Jacob and Jacques Monod in 1961.

An operon is a group of structural genes transcribed together as a single mRNA, plus the control sequences that switch them. The lac operon has three structural genes: lacZ, which codes for β-galactosidase, the enzyme that hydrolyses lactose into glucose and galactose; lacY, which codes for lactose permease, the transport protein that lets lactose into the cell; and lacA, a transacetylase whose function is not settled. In front of them sit the promoter, where RNA polymerase binds, and the operator, a short sequence between the promoter and the genes.

Describe the drawing

The lac operon drawn as a length of bacterial DNA and animated through one full switching cycle. The regulatory gene lacI sits to the left, then the promoter, the operator, and the three structural genes lacZ, lacY and lacA. With no lactose present the repressor protein sits on the operator and RNA polymerase cannot bind the promoter. Lactose then enters, some is converted to allolactose, and that binds the repressor at a separate site; the repressor changes shape, loses its fit for the operator and drifts away. RNA polymerase moves along and transcribes all three genes as a single messenger RNA. When the lactose has been used up the repressor recovers its shape, binds the operator again and transcription stops.

Nearby, and transcribed at a steady rate, is the regulatory gene lacI. Its product is the lac repressor, a protein with two binding sites: one complementary to the operator, and one complementary to the inducer.

In the absence of lactose, the lac repressor binds to the operator and reduces transcription of the structural genes. When lactose is present, some is converted to allolactose. Allolactose binds to the repressor and changes its conformation, so its affinity for the operator falls. RNA polymerase can then transcribe lacZ, lacY and lacA. High expression also depends on low glucose through the cAMP-CAP system.

The three structural genes are transcribed as one long mRNA, ribosomes translate it, and the cell has all three enzymes within a few minutes. When the lactose has been metabolised, allolactose concentration falls, the repressor returns to its original conformation, it rebinds the operator, and transcription falls back to its low background rate.

Two points of precision. Allolactose is an isomer of lactose, formed by β-galactosidase from the small quantity of enzyme the repressed operon still produces, which is why transcription is never entirely absent. If a specification uses the simplified term 'lactose', state that convention in the answer. State the effect of the bound repressor in the terms the specification uses: it prevents RNA polymerase from binding to the promoter, so the structural genes are not transcribed. The mechanism of the block is still under investigation in the research literature, so the specification wording is the one to give.

The cAMP-CAP system is the second arm of the circuit and is not examined at A level in the same detail. Transcription runs strongly only when glucose is also scarce, a state signalled by a rise in cyclic AMP, which activates a protein called CAP that helps RNA polymerase bind the promoter. With glucose plentiful, transcription of the operon remains low even in the presence of lactose.

Cambridge International requires a related pair of terms. β-galactosidase is an inducible enzyme: it is synthesised only when its substrate is present, because a derivative of the substrate reduces the repressor's affinity for the operator. A repressible enzyme is regulated in the opposite direction: it is synthesised continuously until the product of its pathway accumulates, at which point the product binds a repressor protein and gives it a conformation that does bind its operator, so transcription falls. Bacterial enzymes that synthesise amino acids are regulated this way, so a cell supplied with an amino acid reduces transcription of the genes for the enzymes that make it. In one case a small molecule inactivates a repressor and in the other it activates one; in both, transcription is matched to the cell's requirement for the enzyme.

Inducible enzyme

An enzyme synthesised only when its substrate is present, because the substrate or a derivative of it inactivates the repressor of its gene, as allolactose does for β-galactosidase.

Repressible enzyme

An enzyme synthesised continuously until the product of its pathway accumulates and activates a repressor, switching transcription of its gene off.

Exam-board note: the lac operon is named in OCR Biology A, Edexcel Biology A and Cambridge International 9700 and is not named by AQA, whose specification requires repressors acting as transcription factors without naming this example.

Oestrogen and the assembly of a transcription factor

Eukaryotes have no operons: genes with related functions are distributed across chromosomes rather than transcribed together. The mechanism of a protein binding DNA and altering the rate at which RNA polymerase transcribes a gene is the same. AQA names oestrogen as the worked example.

Oestrogen is a steroid and is therefore lipid-soluble, which determines the mechanism. It diffuses through the phospholipid bilayer without a channel or a carrier, so it reaches the inside of the cell and then the inside of the nucleus. A peptide hormone such as insulin can do none of that: it binds a receptor on the outside of the membrane and sends its message in by a second messenger.

Describe the drawing

The path a molecule of oestrogen takes to switch a gene on. It is drawn as an amber disc outside the cell, passing through the two lines of the phospholipid bilayer because it is lipid-soluble. Inside the cytoplasm it binds a receptor protein, drawn as a labelled block, and the two together form a hormone-receptor complex. An arrow carries that complex into the nucleus, drawn as a large circle, where it settles on the promoter region lying beside the target gene and transcription of that gene starts.

Inside a target cell, oestrogen binds to an oestrogen receptor, a protein that is itself an inactive transcription factor. Binding changes the receptor's tertiary structure and exposes its DNA-binding site. The hormone-receptor complex then binds to a specific base sequence beside the target gene, the oestrogen response element, and recruits RNA polymerase, so transcription of that gene starts or speeds up.

A convention to note: textbook diagrams and specification wording place the receptor in the cytoplasm, while most oestrogen receptor protein is in the nucleus before the hormone arrives. Give the cytoplasmic version where a question asks for the A-level pathway.

Only cells that make the receptor can respond to oestrogen at all, which is why the hormone reshapes receptor-rich breast and uterine tissue while tissues carrying few or no receptors barely respond, and why about three quarters of breast cancers are oestrogen-receptor positive and are treated with drugs such as tamoxifen, which occupies the hormone-binding site so the complex cannot form.

Cambridge International names a plant example that works by removing a repressor rather than by activating a factor. The genes for stem elongation and, in germinating barley, for amylase synthesis are repressed by DELLA proteins, which bind the transcription factors for those genes and prevent them acting. The hormone gibberellin binds its receptor, the complex attaches to the DELLA protein, and the DELLA protein, now tagged, is degraded by the cell's protein-degrading system. The transcription factors it was holding are then released, transcription increases, and the stem elongates. Several dwarf wheat varieties carry DELLA proteins that resist that degradation, so transcription is not de-repressed and the stems stay short; those varieties were central to the Green Revolution.

Oestrogen response element

A specific base sequence near a target gene to which an oestrogen-receptor complex binds, increasing the rate of transcription.

Steroid hormone

A lipid-soluble hormone that crosses the cell-surface membrane by simple diffusion and acts on an intracellular receptor.

Epigenetic control of transcription

Two metres of DNA fit inside a nucleus six micrometres across by being wound around histones, positively charged proteins that the negatively charged phosphate groups of the backbone stick to, and then coiled again and again. DNA wound tightly onto histones cannot be reached by RNA polymerase or by transcription factors, so how tightly it is wound is a control mechanism in its own right.

Histone acetylation is the first mark. Enzymes add acetyl groups to lysine residues on the histones' protruding tails, removing the positive charge from each lysine, so the tail's attraction to the DNA's phosphate groups weakens, the chromatin loosens and the promoter becomes accessible. Transcription goes up. Histone deacetylase enzymes remove the acetyl groups, the charge returns and the chromatin condenses. Decreased acetylation means decreased transcription. Like the methylation statement below, that describes the usual effect at a promoter, which is the case the specifications cover.

DNA methylation is the second. A methyl group is attached to a cytosine base, usually where a cytosine is followed by a guanine. Heavy methylation of a promoter silences the gene, partly by preventing transcription factors binding and partly by attracting proteins that recruit the deacetylases, so the two mechanisms are not independent, and the chromatin ends up shut either way.

Describe the drawing

Two panels comparing the same stretch of chromatin in its active and its silenced states. On the left the histone proteins, drawn as grey discs, are spaced well apart with acetyl groups marked on them and the DNA looping loosely between; the promoter is reachable and the gene is transcribed. On the right the same histones are packed tightly together with no acetyl groups, methyl groups are marked on the DNA itself, and the coil is drawn compressed so that nothing can reach the promoter. A line beneath states that neither mark changes the base sequence.

Epigenetics

Heritable changes in gene expression caused by chemical modifications to DNA and to the histones associated with it, without any change to the base sequence.

Epigenome

The full set of chemical marks on a cell's DNA and histones at a given time.

Histone acetylation

The addition of acetyl groups to histone tails, which reduces their positive charge, loosens the chromatin and increases transcription.

DNA methylation

The addition of methyl groups to cytosine bases; heavy methylation of a promoter reduces or prevents transcription of that gene.

The word 'heritable' in that definition covers two distinct claims, which are supported by different amounts of evidence.

Heritable through cell division is well established. When a cell replicates its DNA an enzyme copies the methylation pattern from the parent strand onto the new one, so a liver cell divides into two liver cells rather than into something undecided. That is how a differentiated cell stays differentiated through hundreds of divisions, and it is why the marks are called heritable at all.

Heritable through the germ line, meaning passed to a child in the gamete, is a different claim. Mammalian marks are erased twice: once in the primordial germ cells as gametes form, and again in the zygote shortly after fertilisation. Both erasures are extensive, so most marks a parent acquires during life cannot reach the child. A few regions escape. Imprinted genes, where whether an allele is expressed depends on which parent it came from, are the clear case, and their marks are erased and reset each generation according to the sex of the parent, so they do not accumulate across generations.

Three lines of evidence are relevant. In mice a handful of loci escape erasure, and a mother's diet can change her offspring's coat colour through methylation of one of them. In humans the evidence is observational: children conceived during the Dutch Hunger Winter of 1944 to 1945 show altered methylation at some loci decades later, and records from Överkalix in northern Sweden suggest associations between grandparents' food supply and grandchildren's health. None of that demonstrates a mechanism. A fetus in a starved uterus is directly exposed, other explanations were not excluded, and no molecular route from a grandparent's diet to a grandchild's epigenome has been shown in humans.

The defensible statement is therefore: epigenetic marks are reliably passed from a cell to its daughter cells, and that is how differentiation persists; whether experiences in one human generation routinely leave marks in the next is not established. Note also that the marks are attached to the DNA and its histones rather than altering the base sequence, so no gene is rewritten in either case.

RNA interference: post-transcriptional control

The mechanisms above act before or during transcription. RNA interference acts afterwards, by degrading an mRNA that has already been transcribed, so protein synthesis stops without any change to transcription.

Double-stranded RNA in the cytoplasm, whether from a virus, a transcribed repeat or a laboratory, is cut by an enzyme into fragments about twenty-one nucleotides long: small interfering RNA, or siRNA. One strand of each fragment is loaded into a protein complex and the other discarded. The complex then uses its single strand as a search template, and wherever it meets an mRNA whose bases are complementary it pairs with it and cuts it, leaving the pieces to cytoplasmic enzymes. The gene was transcribed; no protein is made.

Cells also transcribe their own short RNAs, microRNA, from their own genes. In animals these usually bind imperfectly, so instead of cutting the mRNA they slow its translation and speed its breakdown, and the effect is a reduction in the rate of protein synthesis rather than its abolition. One microRNA can regulate hundreds of different mRNAs.

siRNA

Small interfering RNA: short double-stranded RNA fragments, one strand of which guides a protein complex to a complementary mRNA so that the mRNA is broken down and cannot be translated.

RNA interference

The inhibition of gene expression by small RNA molecules binding to complementary mRNA.

Two applications. In the laboratory, siRNA is used to determine the function of a gene by silencing it and recording the consequences. In medicine, siRNA drugs are licensed: patisiran, licensed in 2018, silences transthyretin mRNA, faulty and normal alike, in patients whose faulty protein forms damaging deposits. The basis of the approach is specificity: a conventional drug binds a protein and can bind others of similar shape, while an siRNA is complementary to about twenty-one bases of sequence.

Exam-board note. AQA names RNA interference and requires its effect on translation; Edexcel Biology B lists non-coding RNA among the epigenetic modifications that change gene expression; OCR Biology A, Edexcel Biology A and Cambridge International 9700 do not list RNA interference.