26 Model of Gene Regulation

Throughout the course we have been creating and using models to aid our learning and explanations of complex biological processes. These models are based on scientific observations and must be modified as new information becomes available. Models are simplified to focus on key ideas, but have enough information that they can be used to make predictions. Lastly, our comprehensive models created at the end of each unit have provided examples of how models can be used to produce new medicines and technologies. In the Model of Protein Engineering, we modeled how the amino acids at the enzyme active site could be modified to bind a new substrate and carry out a new chemical reaction. In the Model of Energy and Matter Transformations, we modeled how the metabolism of bacteria could be modified to cause the bacterial to produce a biofuel from cellulose. In the Model of Ras and Cancer, we modeled how a specific Ras mutation caused cancer. Importantly, we also modeled how a drug that binds to mutant Ras could be an effective cancer therapy, a drug that was developed by studying structural models of mutant Ras.

These prior models have focused on recent scientific breakthroughs, but for the upcoming model, we will revisit a 20^th century problem that relates to the focus of the previous chapter, gene regulation in bacteria: how can we use bacteria to produce proteins for therapeutic purposes? Specifically, scientists were curious if bacteria could be used to make the human protein insulin, to treat individuals with type 1 diabetes. Insulin is a protein that is encoded by a gene that is not present in bacteria, but what if the scientists could add the DNA to make insulin into the bacteria? Additionally, how could scientists ensure that the gene was expressed in bacteria, which regulate gene expression differently than eukaryotes? The solution was to modify a familiar system—the lac operon—to create a gene expression system in bacteria that would cause the bacteria to produce a human protein. Here, we will expand on our models showing regulation of lac operon under different conditions, then explore how the operon can be engineered to express insulin under a variety of conditions.

And, in case you were wondering, scientists were successful in using bacteria to produce insulin, and a similar process is used to this day to make insulin for millions of diabetics. Furthermore, the success of this endeavor launched an entire new industry, biotech, that continues to develop increasingly sophisticated treatments for cancer, autoimmune immune disorders, infectious disease treatments, and more.

Chapter Outline

Section 26.1 Sugars and lac Operon Expression

Section 26.2 Insulin and Type 1 Diabetes

Section 26.3 Engineering the lac Operon

Section 26.1 Sugars and lac Operon Expression

In Chapter 25, we learned that E. coli bacteria can metabolize glucose as an energy source by default, and that expression of the lac operon allows bacteria to metabolize lactose as a food source when glucose is not available. But how can you tell when bacteria are using a molecule as a food source? Well, unlike the complicated signaling required for eukaryotic cell division, bacteria will undergo cell division so long as there is an energy source. If bacteria are cultured with glucose, the number of cells increases over time due to cell division by binary fission, until the food source is used up (Figure 26.1, solid line). The energy derived from glucose is used to make ATP in cellular respiration, then the ATP is used to drive the many anabolic synthesis reactions that are part of cell division, including transcription, translation, and DNA replication, and the synthesis of building blocks for new macromolecules.

If E. coli bacteria are instead cultured in media containing only lactose, the number of cells also increases steadily over time, indicating bacteria can also use lactose as an energy source. Let’s review some ideas from Chapter 25 to explain how this works. The lac operon consists of three genes and a regulatory region. The lac operon genes encode proteins for lactose transport (permease, encoded by the lacY gene) and lactose breakdown (beta-galactosidase, encoded by the lacZ gene) (Figure 26.2).

The lac operon regulatory region contains the CRP binding site (CBS), the Plac promoter, and the lacO operator. The regulators of the lac operon bind to specific regulatory sequences under certain conditions: LacI binds to lacO when no lactose is present and represses transcription, whereas CRP binds to the CBS after binding to cAMP, which is present when glucose is not present, to activate transcription. RNA polymerase can only bind effectively to the Plac promoter when CRP is bound and LacI is not. Because LacI and CRP change shape when bound to lactose and cAMP, respectively, these small molecules are allosteric regulators. Note that glucose, while relevant for lac operon expression, does not directly bind to any proteins that regulate the lac operon.

Under conditions when only lactose is present, the LacI repressor binds to lactose and not the lacO operator. Meanwhile the absence of glucose leads to production of cAMP, which binds to the CRP activator and then the complex binds to the CBS. The binding of the activator and the unbinding of the repressor together bring lac operon transcription to maximal levels (Figure 26.3a). Under these conditions, lactose import into the cell is higher due to permease function, and beta-galactosidase is converting lactose to glucose and galactose (see Figure 26.3b for the chemical reaction). Altogether, because the cells can make ATP, rapid cell division occurs until the lactose is used up.

Now let’s consider a new circumstance: E. coli bacteria are cultured with both glucose and lactose. As shown in Figure 26.4a, bacterial cell number increases during time range A, so an energy source is present. Both sugars are present, but which is utilized at time A?

Recall from Chapter 25 that glucose is preferred over lactose as an energy source. This is because the genes needed to metabolize glucose are housekeeping genes that are always expressed, while lac operon expression is regulated. Let’s consider each lac operon regulator in turn to understand why, starting with LacI. Because lactose is present, LacI does not bind to lacO. CRP, meanwhile, is regulated by cAMP levels, and cAMP is not present because glucose is present. Thus, CRP does not have the correct shape to bind to the CBS to activate transcription (Figure 26.4b). What does this mean for RNA polymerase? The repressor is not repressing transcription, but the activator is not activating transcription, so RNA polymerase does not effectively bind to the Plac promoter. Use this information to sketch out a model of the lac operon during time range A, and compare with the model in Figure 26.3a.

While some very low levels of transcription occur (referred to as basal transcription), the amount of protein produced is insufficient to use lactose as an energy source, and bacteria are using glucose for energy during time range A. Over time, this means that glucose levels decrease until no more remains (Figure 26.5a, dashed red line).

This coincides with a lack of new E. coli cells being created for a short period. At the time range marked B, E. coli cell number increases once again (Figure 26.5a). What happened between times A and B? Let’s return to our model of the lac operon. At time B, only lactose is present (Figure 26.5b), and lactose binds to LacI, so LacI does not bind to lacO. However, glucose is no longer present, so the cells produce cAMP, which binds to CRP. Now, CRP can bind to the CBS and recruit RNA polymerase for lac operon transcription to occur. The “lag” in growth between A and B represents the time needed for cAMP to increase, for transcription and translation of beta-galatosidase and permase to occur, for these proteins to start carrying out their functions. We can see that cell division resumes during time range B, indicating that after the lag, the cells are able to effectively use lactose for energy (Figure 26.5a).

In order to make ATP using the energy from lactose, the lactose is broken down into glucose and galactose sugars (Figure 26.3a). Thus, over time, lactose levels also decline (Figure 26.6a, thick dotted line).

When the lactose is used up, cell numbers stop increasing. Why do you think this is? Using your knowledge of the lac operon regulators, try to predict what the lac operon looks like at time range C. Sketch out your model and write an explanation for the shape and function of LacI, CRP, and RNA polymerase.

Section 26.2 Insulin and Type 1 Diabetes

As mentioned at the start of this chapter, scientists wanted to make bacteria that would express insulin protein to treat diabetes. While many eukaryotes produce some form of insulin, scientists were most interested in the version produced by the pancreas of large mammals. In your body, the insulin gene is present in all of your cells, but only certain cells of the pancreas, an organ located near the stomach, activate expression of this gene and produce the protein. After a meal that includes carbohydrates, your blood sugar rises, and this is sensed by the pancreas, which releases insulin into the blood stream (Figure 26.7).

In response to insulin signaling, muscle and adipose (fat) cells import glucose from the blood and convert glucose to energy storage molecules such as the polysaccharide glycogen (muscle), or the lipid triacylglycerol (adipose tissue) (see Chapter 3 for the structures of these macromolecules). Insulin also increases the conversion of glucose to glycogen in liver cells and inhibits the breakdown of glycogen and triacylglycerol in these tissues.

Individuals with type 1 diabetes do not produce insulin, most often due to an autoimmune attack that destroys the specific cells of the pancreas that produce insulin. As a result, these individuals have elevated blood glucose levels that are fatal if untreated. Unfortunately, the link between eating and high blood glucose meant that prior to the discovery of insulin, the only way to survive was by adopting a “starvation diet,” which was understandably hard to stick to. Thus, it was an enormous breakthrough when insulin purified from the pancreases of cows was shown to lower the blood glucose levels of human patients. To facilitate the mass production of insulin, the researchers shared their findings with a pharmaceutical company, Eli Lilly, which began isolating large amounts of insulin from both cows and pigs for therapeutic purposes, sold as “Iletin” (Figure 26.8a and b).

While many diabetics benefited from this insulin therapy, the process had drawbacks. The supply from slaughterhouses could be unreliable. Both pig and bovine insulin are slightly different in their amino acid sequence from human insulin, which sometimes caused allergies. Additionally, there were fears that the need for insulin might outgrow the supply. Finally, the process was laborious and required literal tons of material to produce a small amount of purified insulin. All of this changed in the late 1970s with the development of a process to use bacteria to produce human insulin. After successful clinical trials conducted a few years later, the Food and Drug Administration approved the drug Humulin R, the first “recombinant” insulin, produced by Lilly (Figure 26.8c). This was not only another breakthrough for diabetes care, but it was also the beginning of a new approach for introducing DNA from another species into another living organisms to produce biologic molecules for therapeutic purposes.

Section 26.3 Engineering the lac Operon

As noted above, scientists anticipated that for bacteria to express a human gene, the correct sequences would need to be provided so bacteria would recognize the sequence as a gene to be expressed. At a minimum, a sequence that bacteria would recognize as a promoter would need to be present, so RNA polymerase can bind and start transcription. As summarized in Figure 26.9, human regulatory sequences are structured differently than bacteria sequences. This is because the proteins that interact with those sequences, while similar in overall function, have different structures and therefore different DNA sequences that they recognize.

The safest approach, therefore, was to use sequences known to be recognized by bacteria to regulate expression—the lac operon regulatory sequences. Scientists created a DNA molecule with the insulin gene inserted after the lac operon regulatory sequences. This modified DNA molecule was introduced into bacteria that still have the normal lac operon in their genome. Based on what you know about lac operon regulation, what conditions would lead to insulin expression in these modified bacteria?

The expression of the lac operon is restricted to certain conditions because of the regulatory sequences and the proteins that interact with them—LacI, CRP, and RNA polymerase. When creating an engineered sequence, though, one can change the specific sequences to achieve a desired outcome. For example, after creating bacteria that express typical human insulin, researchers modified the insulin gene sequence to create new insulin proteins with different properties, such as insulin that is longer-acting or shorter-acting. But one could also think about changing how insulin expression is regulated. Some regulatory sequences are modular, meaning you can add, delete, and mix-and-match sequences as needed. Putting on your engineer’s cap, what change could you make to cause insulin expression to occur under different conditions? What if you wanted to make insulin expression constitutive?

Modern genetic engineering involves similar principles, and research labs continue to employ the ideas and techniques developed to create insulin-producing bacteria back in the 1970s, particularly the manipulation of the lac operon. For example, when scientists introduce new DNA into bacteria, they might use the bacterial lacZ gene to track whether they’ve been successful. In Figure 26.10a, the expression of lacZ means the expression of beta-galactosidase enzymes that convert a colorless molecule into a blue pigment.

In my PhD research, I also used lacZ as an indicator as I searched for sequences in the human genome that cause expression of genes in a specific cell type in the brain called serotonin neurons. In some ways, this was a multi-organism experiment, with human sequences connected to the bacteria lacZ gene, then inserted into mice that I could study. Once again, blue color is a marker of lacZ expression, and the blue color you see in the mouse embryo in Figure 26.10b is exactly outlining the developing serotonin system. Finally, if you find yourself in a research lab trying to coax bacteria to produce a protein for you, odds are good that your tool of choice will be the lac operon!