25 Regulation of Gene Expression II: Bacterial Gene Regulation

In the previous chapter, we explored the breadth of gene regulation that occurs in eukaryotic cells like our own. Now to explore some specific gene regulation pathways in depth, we will focus on some very well-studied gene regulation systems from bacteria. Gene regulation is especially important for single-celled organisms; bacteria are optimized for growth, and energy expended on gene expression is not available for other cellular functions. After we investigate how bacteria regulate their gene expression (this chapter), we will see how this knowledge was used to produce human insulin in bacteria (Chapter 26), a breakthrough that continues to be a vital therapeutic for individuals with type 1 diabetes.

Chapter Outline

Section 25.1 Comparing Eukaryotic and Bacterial Genes and Regulation

Section 25.2 The Bacterial trp Operon

Section 25.3 The Bacterial lac Operon

Section 25.1 Comparing Eukaryotic and Bacterial Genes and Regulation

Eukaryotes and bacteria comprise two major forms of life, and while this text emphasizes broad principles that apply to all forms of life, we have discussed some key differences in cellular structures and processes that are specific to cells of each type. On the topic of gene regulation specifically, the different organization of eukaryotic versus bacteria cells directly impacts gene regulation processes. For example, eukaryotic cells house their genomic DNA in the nucleus, so that is where transcription occurs (Figure 25.1).

As discussed in Section 24.3, following transcription, the primary transcript is modified to produce the mRNA that can leave the nucleus to be translated in the cytosol, where ribosomes are located. In contrast, bacterial cells house DNA and ribosomes in the same cellular compartment, the cytosol. Bacterial RNAs do not undergo the extensive modifications observed in eukaryotes because they do not need to be transported to another compartment, and thus can be immediately translated by a ribosome. Indeed, the processes of transcription and translation can occur in tandem, with translation of the mRNA initiating even before transcription is complete! Thus, while bacteria can rapidly produce new proteins, the fine-tuning of eukaryotic gene expression and alternative splicing that we learned about in Chapter 24 do not occur in bacteria.

Another key difference between eukaryotic and bacterial gene regulation relates to the organization of genes. In the eukaryotic gene model, each gene is controlled by one regulatory region, which may consist of multiple enhancers and silencers in addition to the promoter (Figure 25.2a). We also saw that one kind of primary transcript was produced, often containing several exons and introns, and while different splicing could produce different mRNAs, once the mRNA is made, only one type of protein is produced in translation.

By contrast, some bacterial genes are grouped together into operons, in which a single regulatory region controls expression of several genes (Figure 25.2b). In most cases, these genes code for proteins that carry out related processes in the cell, so their coordinated expression is useful. When the operon is expressed, one type of mRNA is produced with the coding sequences corresponding to each gene in the operon. Each coding sequence has its own start and stop codons, such that translation of the mRNA produces one type of protein for each gene. Therefore, one operon contains multiple genes, and one type of mRNA produces multiple proteins when translated.

As shown in Figure 25.2b, the names and order of regulatory sequences also differ between eukaryotes and bacterial, perhaps because they were studied by different scientists. However, there are many similarities despite the similar terms. Importantly, while expression of many eukaryotic genes requires many different general transcription factors to assemble at the promoter (see Figure 24.7), bacterial RNA polymerase has a high affinity for some promoter sequences and can bind without assistance (Figure 25.3a).

In this case, expression of genes occurs by default, but RNA polymerase binding can be prevented by a repressor binding to an operator sequence, which is analogous to the negative transcription factor binding to a silencer in eukaryotic genes. The operator sequence is typically between the promoter and coding sequences, and you can imagine that when the repressor protein binds to the operator, this physically blocks the binding of RNA polymerase and prevents the reading of DNA template strand. This form of gene regulation is referred to as negative transcriptional regulation, since a negative regulator (repressor) must bind to inhibit transcription. For other genes, RNA polymerase has a low affinity for promoter sequences, and an activator protein must bind first to a sequence adjacent to the promoter, called the activator binding site. The binding of activator protein to its binding site typically changes the shape of the promoter DNA sequence to make it more accessible to RNA polymerase, and transcription can now occur. The function of the activator protein in the latter example, also called positive transcriptional regulation, is analogous to a positive transcription factor binding to an enhancer sequence in eukaryotic systems.

As we delve into specific examples in the next sections, it may help to create a list of key proteins and corresponding DNA sequences for each scenario. Additionally, try to identify whether negative regulation, positive regulation, or some combination of the two is occurring. Finally, keep in mind the functions of the genes in each operon, and try to relate these functions to how the operon is regulated.

Section 25.2 The Bacterial trp Operon

All cells need a constant supply of amino acids, as protein synthesis is always occurring in cells. Our cells can make many amino acids, some must be supplied by our diet. Tryptophan is one of nine such essential amino acids. However, an E. coli bacterium can either obtain tryptophan from the environment, or produce its own supply. The tryptophan (trp) operon codes for five enzymes that act in a pathway to produce tryptophan from a precursor molecule (Figure 25.4a). Since gene expression expends energy, bacteria benefit by expressing the trp operon only when it is needed, i.e., when tryptophan is not available in the environment. However, if tryptophan supplies are plentiful (think about your gut after a large, protein-rich meal), bacteria can spend their energy on processes other than making tryptophan.

The regulatory region of the trp operon consists of two components: a promoter (Ptrp) and an operator (trpO). The regulator of the trp operon is the Trp Repressor (TrpR), which can bind to the trpO operator to block transcription. Tryptophan is itself is the regulator of the TrpR repressor: when tryptophan is present, it binds to the TrpR repressor, changing its shape so that TrpR can bind to the trpO operator (Figure 25.4b). However, when tryptophan is absent, the TrpR repressor has a different shape that does not allow for DNA binding. Because RNA polymerase has a high affinity for the Ptrp promoter, transcription occurs so long as the repressor is not bound (Figure 25.4c). If the environment changes and tryptophan levels increase, the TrpR repressor binds to the trpO operator to block trp operon expression. This is an example of negative transcriptional regulation, in which expression occurs until prevented by the repressor.

In Figure 25.4c, notice that RNA polymerase and the TrpR repressor are present in the cell, even though only one of them is carrying out its function. RNA polymerase enzymes are present in cells because transcription of genes is always occurring. Similarly, the TrpR repressor is always present in these cells to allow for dynamic changes to trp operon expression based on levels of tryptophan. Thus, you should always include RNA polymerase and TrpR repressor in your models of the trp operon, but draw these as clearly bound to DNA or not as appropriate for the tryptophan environment. And although these are not shown in our models, we know that genes code for proteins, so somewhere else in the genome are the genes for TrpR and proteins of the RNA polymerase complex. These are examples of housekeeping genes, or genes for proteins that always need to be expressed to carry out essential functions in the cell. Another word for this is constitutive—always being transcribed and translated. What other genes can you think of that might be constitutive, either in bacteria or in eukaryotic cells (hint: think of essential processes that are always occurring in cells!)?

Section 25.3 The Bacterial lac Operon

In parallel with their ability to produce all amino acids needed to make proteins, E. coli bacteria have also developed the ability to utilize many diverse molecules as food sources. However, E. coli bacteria have preferred foods, as I’m sure you do too. If E. coli bacteria are presented with several types of sugar molecules, the bacteria will use glucose first. This is because E. coli bacteria already express the genes for glucose breakdown—the genes that code for glycolysis enzymes and other proteins needed for glucose oxidation are housekeeping genes. To utilize other sugars, such as the disaccharide lactose, new gene expression must take place to produce the proteins required for lactose metabolism. The lactose (lac) operon codes for these genes, and the regulation of the lac operon ensures that substantive expression of the operon only occurs when two conditions are met: glucose is NOT available, and lactose IS available.

The lac operon is different from the trp operon in two key ways. First, trp operon expression is influenced by one molecule, tryptophan, while lac operon expression requires specific levels of two sugars, glucose and lactose. This makes lac operon regulation at least twice as complicated as the trp operon. Furthermore, while trp operon genes encode enzymes to synthesize tryptophan, the lac operon genes facilitate the use of lactose as an energy source. In terms of regulation, bacteria benefit from expressing the trp operon mainly when tryptophan is absent, while bacteria benefit from lac operon expression only when lactose is present. As we explore the details of lac operon components and regulation, you may find it helpful to revisit this big picture perspective.

The lac operon has three genes: lacZ, lacY, and lacA (Figure 25.5a). The lacZ gene codes for the enzyme beta-galactosidase, which cleaves the disaccharide lactose into the simple sugars glucose and galactose, which are then quickly metabolized by glycolysis or related pathways. The lacY gene codes for lactose permease, a plasma membrane transporter that increases the transport of lactose into the cell where beta-galactosidase can break it down. Finally, lacA encodes a transacetylase enzyme, whose functions are not directly regulated to lactose metabolism, so we will not discuss this enzyme further.

To ensure lac operon expression is responsive to two molecules, two separate proteins serve as regulators of the lac operon (Figure 25.5b). A repressor called LacI (lac inhibitor) is sensitive to lactose levels. When present, lactose binds to LacI, changing the protein’s shape so LacI does not bind to DNA. However, when lactose is absent, LacI binds to the lac operator, lacO, which blocks transcription of the operon (Figure 25.6a). The actions of the LacI repressor are important to prevent transcription of lac operon genes when there is no lactose to break down.

The other key regulator of lac operon expression is the cAMP receptor protein (CRP). CRP does not bind to glucose, but is indirectly sensitive to glucose levels—when glucose is low, bacteria cells convert ATP to cAMP, and cAMP binds to CRP. The cAMP-CRP complex has a shape that allows for binding to the CRP binding site (CBS) in the lac operon regulatory region. When bound to DNA, cAMP-CRP acts as an activator, changing the shape of the Plac promoter sequence so that RNA polymerase can effectively bind and transcribe the operon. When glucose is high (as in Figure 25.6a), no cAMP is produced, so CRP remains inactive and is not bound to DNA. In the absence of activator binding, RNA polymerase has low affinity for Plac, which means only very small amounts of lac operon transcription occur in the presence of glucose and lactose. Thus, bacteria do not express the lac operon if a readily digestible food source (glucose) is still available. Maximal expression of the lac operon only occurs when the LacI repressor is not bound to DNA and the activator CRP is bound to DNA (Figure 25.6b), which corresponds to the conditions when lactose is present and glucose is absent. Both positive and negative transcriptional regulation are part of lac operon regulation.

Like the trp operon, the LacI and CRP proteins that regulate the lac operon are always present in cells, but toggle between shapes that are active (bound to DNA) or inactive (not bound to DNA). Because these proteins are always present, we can infer that the genes that encode these proteins are constitutively expressed, housekeeping genes. Consider the role of RNA polymerase in the expression of LacI and CRP—would you predict that RNA polymerase has a high or low affinity of the promoters of the genes for LacI and CRP, and why?