Controlling Genes in Prokaryotes: The lac and trp Operons

Why make what you don't need?

An E. coli cell has about 4300 genes, but it does not need all of their proteins all the time. Making a protein costs a lot of energy (you calculated about four high-energy bonds per amino acid in the translation lesson). A bacterium that makes lactose-digesting enzymes when there is no lactose around wastes energy that a competitor could use to grow and divide.

So cells regulate gene expression: they turn genes on and off in response to conditions. Some genes, such as those for ribosome proteins, are needed all the time and are expressed constitutively. Others are switched on only when needed. Expectation D3.3 asks you to explain how regulatory proteins control gene expression in prokaryotes, using operons as the main example.

The operon model

In 1961, François Jacob and Jacques Monod in Paris proposed the operon model; they shared the 1965 Nobel Prize in Physiology or Medicine with André Lwoff. An operon has:

• a promoter, where RNA polymerase binds;
• an operator, a short sequence overlapping or just after the promoter, where a repressor can bind;
• several structural genes for enzymes in one pathway, transcribed together into one mRNA (a polycistronic mRNA, each part with its own start and stop codons).

A separate regulatory gene, with its own promoter, makes the repressor protein. The repressor is an allosteric protein: a small molecule binding to it changes its shape and therefore whether it can bind the operator. (You met allosteric regulation of enzymes in the Biochemistry unit.)

The lac operon: an inducible operon

Lactose (milk sugar) is a disaccharide of glucose and galactose. The lac operon codes for three proteins:

• lacZ: β-galactosidase, which hydrolyses lactose into glucose and galactose (and also converts some lactose to allolactose);
• lacY: permease, a membrane transport protein that brings lactose into the cell;
• lacA: transacetylase, whose role is less important.

No lactose: OFF. The regulatory gene lacI is always transcribed and makes the lac repressor. The repressor binds the operator, blocking RNA polymerase. (A few molecules of the enzymes are still made occasionally, which lets a little lactose in when it appears.)

Lactose present: ON. Some lactose is converted to allolactose, the inducer. Allolactose binds the repressor at an allosteric site, the repressor changes shape and can no longer bind the operator, and RNA polymerase transcribes the three genes. When the lactose is used up, allolactose leaves the repressor, the repressor binds the operator again, and the operon shuts off.

Because the repressor turns the operon off, this is negative control. Because the operon is normally off and is switched on by a nutrient, it is inducible, which makes sense for an enzyme that breaks down a food.

Glucose comes first: positive control by CAP

E. coli prefers glucose, which needs no extra enzymes. If both sugars are present, the cell uses glucose first. This works through a second, positive control:

• When glucose is low, the cell's level of cyclic AMP (cAMP) rises.
• cAMP binds the catabolite activator protein (CAP).
• CAP-cAMP binds a site just upstream of the lac promoter and helps RNA polymerase bind, increasing transcription many times over.
• When glucose is high, cAMP is low, CAP does not bind, and transcription stays low even if lactose is present.

So the lac operon is strongly on only when lactose is present AND glucose is absent.

The trp operon: a repressible operon

E. coli needs the amino acid tryptophan to make proteins. If tryptophan is in its surroundings, it saves energy by not making its own. The trp operon contains five genes (trpE, trpD, trpC, trpB, trpA) coding for enzymes that build tryptophan.

• The regulatory gene trpR makes a repressor that is inactive on its own. It cannot bind the operator, so the operon is normally ON.
• When tryptophan is plentiful, tryptophan binds the repressor as a corepressor. The repressor changes shape, becomes active, binds the operator, and transcription stops.
• When tryptophan runs low, it leaves the repressor, the repressor falls off, and the genes are transcribed again.

This is negative control (a repressor turns it off) of a repressible operon. Repressible operons are typical of pathways that make a product: the product itself shuts down its own production, a form of negative feedback.

The trp operon also has a second, finer control called attenuation, discovered by Charles Yanofsky: when tryptophan is high, the RNA folds in a way that ends transcription early. Attenuation is possible because bacteria translate mRNA while it is still being transcribed.

Operons at work in biotechnology

Biotechnologists borrow the lac system to switch on engineered genes. They place a gene of interest (for example, a human protein) after a lac promoter and operator in a bacterium. To turn it on, they add IPTG, a molecule that binds the lac repressor like allolactose but is not broken down, so the gene stays on at a steady level. The lacZ gene is also used as a marker in cloning (you will see "blue-white screening" in The Biotechnology Toolkit).