More on Operons

Last week I finally addressed promoters and gave a bit of background on the lac operon. Looking back, I feel that I didn't give the topic of operons adequate treatment. They are a very crucial aspect of bacterial genetics and also important for synthetic biology. Many of the systems that synthetic biologists construct include one or more operons. We are often interested in regulating the production of a group of protein products with a particular factor (click image to see a larger version).

It turns out that the lac operon is more highly regulated than what my last post led you to believe. I explained that when lactose is absent, a protein—the lac repressor, encoded by the lacI gene—binds to the operator, blocking RNA polymerase from transcribing the genes of the operon into mRNA. However, if lactose is introduced it binds to the lac repressor, causing it to fall off the operator and clearing the path for RNA polymerases. I called this system inducible, because it is shut off by default but switched on if lactose is added.

This is an incomplete and, admittedly, a misleading picture. In reality, lactose cannot bind to the lac repressor. Instead what happens is that lactose is modified into an isomer, allolactose, which is able to bind to the lac repressor. The lactose is able to be imported into the cell because the permease protein encoded by the lac operon is present at a low level when no lactose is available. In other words, the lac promoter is leaky; the proteins of the lac operon are produced at a low background level.

Last time I omitted another detail that is actually rather important. Not all food sources are equally appealing to E. coli. Glucose is more preferable than lactose because E. coli has to expend energy to convert lactose into glucose before it is usable. Clearly individuals that utilize glucose instead of lactose when both are plentiful will be rewarded. Evolution has produced a mechanism that accomplishes this. The lac operon contains what is known as the CAP site, located upstream of the promoter along the DNA. The CAP site gets its name from a protein-the catabolite activator protein-that can bind there. When glucose is plentiful, the CAP site is vacant. However, when glucose is absent an enzyme called adenylyl cyclase is active. Adenylyl cyclase produces a molecule called cAMP (pronounced "cyclic A-M-P"). When the level of cAMP is high, some molecules of cAMP bind to CAP, which allows the CAP-cAMP complex to bind to the CAP site of the lac operon. When the CAP site is bound, RNA polymerase has a higher affinity for the lac promoter and binds more often. This causes the cell to produce more of the proteins encoded by the lac operon. The details of the lac operon's operation are provided in the image above. I hope its size is not too distracting; I wanted you to be able to read the small type!

To summarize, the lac operon is fully turned on only when lactose is present and glucose is absent. Combined, these two conditions increase E. coli's metabolic efficiency, so they have been favored by natural selection. The lac operon is dubbed inducible because it is off by default, but switched on when lactose-the inducer-is present. It is also said to be negatively controlled, because a repressor protein is bound to the operon by default, shutting it off. This is not the only strategy for gene regulation. Some operons are repressible instead of inducible, meaning that the presence of a small molecule-called a corepressor-shuts the operon off. Operons also differ by their control-positive or negative. Operons under positive control are switched on when a protein is bound to the operator, whereas operons under negative control are switched off.

A frequently cited example of a repressible operon under negative control is the trp operon, responsible for manufacturing the amino acid tryptophan. Since the proteins of the operon manufacture a product, tryptophan, rather than break something down as is the case for the lac operon, evolution has selected a different regulation scheme. If tryptophan is abundant, then the cell needn't expend energy to manufacture more. Its energy would be better spent on other functions. So, tryptophan acts as a corepressor for its own operon. When tryptophan is absent, the repressor protein is unable to bind to the operator, and the proteins required to synthesize tryptophan are produced. When the level of tryptophan rises, though, it binds to the repressor, allowing the protein to bind to the operator and blocking RNA polymerase from binding.

I have only provided a glimpse of the wide array of mechanisms that have evolved to regulate gene expression. Based on my own observations, promoters and operons alone seem to be the most commonly used method to regulate gene expression in synthetic biology. However, if we are to build artificial systems that match the caliber of those found in nature we will need to learn how to apply all the tools at our disposal.

Image Credit: G3pro & Tereseik (via Wikimedia)

References and Further Reading:

Brown, T. A. Genetics: A Molecular Approach. 2nd ed. London: Chapman & Hall, 1992.

Dale, J. W. Molecular Genetics of Bacteria. Chichester: John Wiley & Sons, 1989.