Gene Expression and Cell Specialization: Study Guide (Biology 6.6)

One genome, many kinds of cell

You started as a single cell. That cell divided by mitosis, so nearly every cell in your body carries the same DNA. Yet a nerve cell, a muscle cell and a pancreatic beta cell look and work very differently. Beta cells make insulin; developing red blood cells make haemoglobin. Each cell type still carries the gene for the other’s protein. It simply does not use it.

This is differential gene expression: different cells express different subsets of the same genome. The proteins a cell makes decide its structure and its job, so gene regulation is what turns one genome into many cell types. In Topic 6.5 you saw that regulatory proteins bind regulatory DNA sequences. This topic shows how that works in eukaryotic cells, and adds a second layer of control by small RNA molecules.

Starting transcription at a promoter

In a eukaryotic cell, RNA polymerase cannot find a gene on its own. It needs helper proteins called transcription factors.

1. The promoter is a DNA sequence just upstream of the transcription start site. Many promoters include a short sequence rich in T and A, the TATA box, roughly 25 to 35 base pairs before the start site.
2. General transcription factors bind the promoter first. They are needed at nearly every gene.
3. RNA polymerase then binds to the promoter–factor complex. Transcription can begin, but on its own this complex starts transcription only rarely. The result is a low, “basal” rate.

High rates of transcription need extra proteins that are specific to particular genes and cell types.

Enhancers and activators: turning transcription up

An enhancer is a regulatory DNA sequence that binds activators, the gene-specific transcription factors. Enhancers have three surprising features:

• They can be far from the gene, sometimes many thousands of base pairs away.
• They can be upstream of the start site, downstream of it, or even inside an intron.
• They still act on their own gene, because DNA is flexible.

How a distant enhancer works. The DNA between the enhancer and the promoter bends into a loop. This brings the activators on the enhancer into contact with a group of mediator proteins, which in turn contact the general transcription factors and RNA polymerase at the promoter (Figure 1). The activators help the whole complex assemble and start transcription much more often. More activator bound means a higher rate of transcription, more mRNA and, usually, more protein.

Repressors: turning transcription down

Regulation also works in the negative direction. Negative regulatory molecules, usually repressor proteins, bind to DNA and block transcription. They can:

• bind a silencer sequence and stop the transcription complex from assembling;
• sit on or next to an enhancer, so activators cannot bind;
• bind near the promoter, so RNA polymerase cannot start.

You met the same idea in the lac operon, where a repressor on the operator blocked RNA polymerase. In eukaryotes, a gene’s control region often has binding sites for both activators and repressors. The balance between them decides the rate of transcription.

Combinations of transcription factors

A human cell uses only a limited number of different activator types, yet it must control thousands of genes in hundreds of cell types. The solution is combinatorial control: each gene’s control region has binding sites for a particular combination of activators, and the gene is switched on strongly only when that whole combination is present.

Here is a model with four activators, A to D, and three genes.

Gene	Activators its enhancer needs
gene 1	A and B
gene 2	A and C
gene 3	B and D

Cell type	Activators present	Genes switched on
cell X	A, B, D	genes 1 and 3
cell Y	A, C	gene 2
cell Z	B, C, D	gene 3

Every cell contains all three genes. Activator A is in both X and Y, but it switches on different genes in each, because its partners differ. A small set of regulators, used in different combinations, gives each cell type its own pattern of gene expression, and so its own products and functions.

From regulation to phenotype

Differential gene expression explains how cells specialise:

• A muscle cell expresses genes for contractile proteins such as actin and myosin at high levels, so it can contract.
• A beta cell in the pancreas expresses the insulin gene, so it can secrete insulin.
• A change in regulation, such as a missing activator or a damaged enhancer, can change how much of a protein is made, or in which cells it is made. This can change the phenotype of a cell, a tissue or the whole organism, even if the protein’s own coding sequence is normal.

Small RNAs: control after transcription

Some genes code for RNA molecules that are never translated but regulate other genes. Two important kinds are short, about 21 to 23 nucleotides long:

• MicroRNAs (miRNAs). A miRNA gene is transcribed into RNA that folds into a hairpin. Enzymes cut out a short single strand, which is held in a protein complex. The miRNA base-pairs with a complementary sequence on target mRNAs. In animals the match is often partial and usually in the 3′ untranslated region. The result is that translation is blocked, or the mRNA is broken down, or both. One miRNA can regulate many different mRNAs.
• Small interfering RNAs (siRNAs). These are cut from long double-stranded RNA, such as the RNA of some viruses. They work in the same protein complex but usually match their target exactly, which leads to the target mRNA being cut and destroyed.

Researchers use this natural system as a tool. Adding double-stranded RNA that matches a chosen gene triggers RNA interference (RNAi), lowering that gene’s expression so its function can be studied. The gene’s DNA is not changed, so the effect wears off as the added RNA is used up.

Level of control	Molecule that regulates	Effect
chromatin (Topic 6.5)	enzymes adding or removing methyl and acetyl groups	gene accessible or packed away
start of transcription	general transcription factors, activators, repressors	rate of mRNA production
after transcription	miRNAs and siRNAs	less translation or faster mRNA breakdown

Worked example 1: finding enhancers with a reporter gene

Question. A fictional mouse gene, Kd1, is expressed strongly in kidney cells but hardly at all in liver cells. Researchers join its control region to a reporter gene that makes a light-emitting enzyme; the amount of light shows how much transcription the control region drives. The control region contains a promoter, a region E1 about 3000 base pairs upstream, a region R about 800 base pairs upstream, and a region E2 inside the first intron, downstream of the start site. Each version, with one or more regions deleted (Δ), is put into kidney cells (light units; data fictional).

Construct	Light units	% of full-length
full-length	1000	100
ΔE1	380	38
ΔE2	450	45
ΔE1 and ΔE2	30	3.0
ΔR	2100	210
Δpromoter	2	0.2

The full-length construct gives 25 units in liver cells.

(a) Identify the role of E1, E2, R and the promoter. Support each claim with data. (b) Explain why the full-length construct gives so much more light in kidney than in liver cells.

(a) Claims and evidence.

1. Promoter: essential. Without it, activity falls to 0.2% (2 ÷ 1000 × 100). RNA polymerase and the general transcription factors have nowhere to assemble.
2. E1 and E2: enhancers. Removing E1 cuts activity to 38%; removing E2 cuts it to 45%. Removing both leaves only 3.0%, near the basal level. So each region raises transcription.
3. The two enhancers work together. If each enhancer multiplied the rate independently, removing both would leave about 0.38 × 0.45 = 0.171, or 17%, of full activity. The measured value, 3.0%, is far lower. Each enhancer is much more effective when the other is present.
4. E2 is downstream. It lies inside an intron, yet it still enhances transcription. The position of an enhancer, upstream or downstream, does not stop it working.
5. R: silencer. Removing R raises activity to 210% (2.1 times full-length). So R normally lowers transcription, most likely by binding a repressor.

(b) Tissue difference. Liver cells give 25 units, only 2.5% of the kidney value, or 1/40. Both cell types received the same DNA. The difference must come from the proteins inside the cells: kidney cells contain the activators that bind E1 and E2; liver cells lack them, or contain more of the repressor that binds R. This is differential gene expression caused by different sets of transcription factors.

Limits. A reporter gene in a cell culture is a model. It shows what the control region can do, but the gene in its normal place on the chromosome may also be affected by chromatin packing.

Worked example 2: supporting a claim about a microRNA

Question. In fictional muscle cells, a microRNA called miR-X is suspected of controlling protein P. Researchers measure P mRNA and P protein, relative to untreated cells, under four conditions.

Condition	P mRNA	P protein	Protein ÷ mRNA
untreated (miR-X present)	1.0	1.0	1.0
miR-X blocked by a complementary RNA	1.9	4.2	2.2
P mRNA made without the miR-X binding site	2.0	4.0	2.0
unrelated control RNA added	1.0	1.05	1.05

Make a claim about how miR-X affects protein P, and support it with evidence and reasoning.

1. Claim. miR-X reduces the amount of protein P by base-pairing with P mRNA. It does this in two ways, to a similar extent: it blocks translation and it causes the mRNA to be broken down.
2. Evidence that miR-X is responsible. Blocking miR-X raises P protein 4.2-fold. The unrelated control RNA has almost no effect (1.05), so the rise is not just a result of adding RNA to the cells.
3. Evidence that it acts on the mRNA itself. Removing the binding site from P mRNA gives almost the same rise (4.0-fold) as blocking miR-X. miR-X needs to pair with that site to work.
4. Evidence for two effects. P mRNA roughly doubles (1.9 and 2.0), so miR-X normally speeds up mRNA breakdown. But protein rises about twice as much as mRNA: protein per mRNA goes from 1.0 to 2.2 (4.2 ÷ 1.9) and 2.0 (4.0 ÷ 2.0). Each mRNA is translated about twice as often when miR-X cannot bind, so miR-X also blocks translation. The two effects are about equal (about 2-fold each), and together they multiply: 1.9 × 2.2 ≈ 4.2.
5. Reasoning. Base-pairing between a small RNA and its target mRNA lowers gene expression after transcription. Muscle cells that make miR-X will therefore have less protein P than cells that do not, which is one way cell types differ even when both transcribe the same gene.

Common misconceptions

• “Different cells have different genes.” Almost all cells have the same genome. They differ in which genes they express.
• “Enhancers must be just upstream of the gene.” They can be far upstream, downstream or inside introns. DNA looping brings them close to the promoter.
• “An activator binds the promoter.” General transcription factors bind the promoter. Activators bind enhancers and act on the promoter complex through mediator proteins.
• “One transcription factor controls one gene.” Most genes need a combination of factors, and one factor can help control many genes.
• “miRNAs change the DNA of the target gene.” They act on mRNA, after transcription. The gene is unchanged.
• “If mRNA level does not change, the protein level cannot change.” Small RNAs can block translation, lowering protein without much change in mRNA.
• “Transcription is either on or off.” Activators and repressors set a rate, which controls how much protein is made.

Where this leads

Changes to DNA sequences, in coding regions or in regulatory sequences like enhancers and promoters, are the subject of Topic 6.7, Mutations. Before moving on, try the practice questions, then use the revision notes and the checklist.