Prokaryotic Gene Control

Edited Notes by Amy Stone and Brianna Dusseault

Operon Theory - Lac Operon

Regulation of a gene usually occurs through transcriptional control. Operon Theory states that genes that function together are often regulated together and are known as an operon. They occur in prokaryotic organisms.

One of the most thoroughly studied operons is the lac operon which was first discovered in E. Coli. E.Coli can choose between many food sources, including lactose and glucose. However, the organism prefers glucose over any other food source. The genes in the lac operon are responsible for lactose metabolism. The three genes that are regulated together in the lac operon control the production of enzymes which convert lactose into simple sugars (including glucose). They are -galactosidase, which breaks down lactose, galactoside permease, which gets lactose into the cell, and galactoside transacetylase, whose function is uncertain.

The operator site is located on the region between the promoter and the first gene of the operon. Further upstream on the DNA lies a lac repressor gene which synthesizes a lac repressor protein. The operator serves as the binding site for the lac repressor protein. When the lac repressor protein binds to the operator, it will prohibit an RNA polymerase from transcribing the lactose gene. This happens because when the lac repressor is bound to the operator, the physical shape of the lac repressor blocks the path of the polymerase.

If we add lactose to the system and remove glucose the cell is in dire need of metabolizing this lactose as an energy source. When lactose is present, it will bind to the repressor and change its shape. The lactose-repressor complex can no longer recognize the operator sequence and does not bind to the DNA. Now the RNA polymerase can move down the DNA and carry out transcription. The restoration of gene expression from the lac operon in this case is an example of de-repression.

A mutation in any of the parts of the operon sequence can result in an imbalance of lactose synthesis. A mutation in the DNA of the operator sequence that prevents the repressor from binding would result in the lac operon genes being always turned on. A mutation in the lac repressor gene that causes it to lose its ability to recognize the operator would also cause the operon to turn on, even in the absence of lactose. This is because the repressor would be incapable of recognizing and binding to the operator. A mutation in a repressor that prohibits the lactose from binding turns the synthesis off even if there is lactose in the medium. If there was a mutation in both the lac repressor gene and the operator region they might compensate for each other and a lactose control could be restored.

Dealing with Glucose

Glucose is the preferred sugar as an energy source for cells. Therefore, if glucose is present, the lactose metabolism needs to be turned off to maintain efficiency. To deal with this contingency there is an activator site in front of the promoter and operator. When a small molecule called cAMP is present in the cell, it will bind to an "activator" protein. When the two combine, the activator protein will bind to the activator site. The activator protein helps to unwind the DNA and make transcription of the lac operon easier. Without the activator protein-cAMP complex, the shape of the activator protein changes and the activator can no longer bind to the activator site. The result is that there will be a poor synthesis from the lac operon. The level of cAMP is affected by levels of glucose in the cell. If the cell has high glucose levels, cAMP leaks out of the cell and creates low intracellular cAMP levels. This means that many of the activator proteins will not pair up with cAMP and thus cannot bind to the activator site on the DNA. This reduces transcription from the lac operon when glucose is in the cell.

The control system utilizing cAMP and the activator protein influences the expression in many operons. For efficiency, the cell prefers to utilize glucose over any other energy source. Thus, high levels of glucose will prevent expression from any operon that metabolizes an alternative energy source. If glucose levels drop (and cAMP levels rise as a consequence), alternative energy sources can be utilized.

Tryptophan (Trp) Operon

The trp operon is responsible for synthesizing the amino acid tryptophan. All organisms need tryptophan in order to live. Here, when tryptophan is present the cell turns the tryptophan synthesis off because there is no need to make more. This is, therefore, opposite from the lac operon.

On the trp operon, a repressor protein that is unable to bind to the trp operator is made. Because it does not bind to the operator, the gene is always "on." When trp becomes more prevalent in the cell, it will bind with the repressor and change its shape so that the repressor can bind to the operator. This stops the synthesis of the trp operon. As the level of trp rises, more and more repressors will be able to bind to the operator and reduce trp production. In this way, the trp operon is an elegant system that can regulate its presence in a cell.

Bacteriophage Use Operons

The makeup of a phage (a virus that infects bacteria) includes a geometrical head that holds phage DNA, a long "tail" that is attached to a straight base plate, and legs that are also attached to the baseplate. When a bacteriophage plans to invade a host, it will first attach itself to the surface of a bacterial cell by anchoring its base plate into the cell wall. The sheath of the phage tail then contracts, puncturing the cell surface and injecting the phage DNA into the cell. The phage DNA contains genes that take over the machinery of the host cell and create hundreds or thousands of progeny phages inside the cell. The cell eventually lyses (breaks open) and the new progeny phages are released into the medium. See figure 4.4 in text.

The phage must be able to regulate the expression of all its genes so that they can take over the cell and create new phages in the right order. For example, the phage must control the lysis gene (which causes the cell to burst) so that it is expressed towards the end of the infective cycle when all of the DNA has replicated and new phages have been assembled. The virus uses operons to regulate gene expression.

T7 Phage

The T7 phage has two promoters - an early promoter and a late promoter. Once the T7 phage has injected its DNA into a host E. Coli cell, the E. Coli RNA polymerase will recognize and bind to the early promoter on the phage DNA. This results in the synthesis from the early genes (the early operon). One of the products of these early stage genes is a new RNA polymerase - the T7 RNA polymerase. The T7 RNA polymerase then binds to the late promoter of the T7 DNA and transcribes the late genes (the late operon). Among the late genes is a lysis gene that causes the host cell to lyse and release the progeny phage. In this way the T7 phage can transcribe its genes and their instructions in a structured order.

T4 Phage

The T4 phage has three operons. As with the T7 phage, the E. Coli RNA polymerase will identify the early promoter on the phage DNA and bind to it. One of the early genes makes a product that degrades the original sigma factor so that it can no longer bind with the RNA polymerase. This stops transcription of the early operon. At the same time, another early gene makes a new sigma factor, which forms a new holoenzyme with the core E. coli RNA polymerase. The new sigma factor instructs the polymerase to transcribe the DNA starting at the middle promoter. A similar process allows the switch to occur again between the middle and late promoters.

Lambda Phage

Control of gene expression in lambda phage utilizes antitermination as well as the mechanisms discussed previously. The lambda phage infects in two ways and is therefore called a temperate phage.

The integrated phage DNA in a lysogenic infection is called a prophage. This lambda DNA lies dormant in the host cell DNA until the right conditions occur for it to turn on its genes. The host cell lives happily until the lambda gets prompted to grow and take over the cell. The lambda prophage now behaves like a time bomb that can be triggered to enter into a lytic infection, just like for the T7 and T4 infections.

The first genes to be turned on in the lambda phage infection are the integration genes, which cause the DNA of lambda to integrate into the host cell's DNA. During the immediate early infection, a product is made which interferes with the host rho factor. Interfering with the rho causes the RNA polymerase to ignore rho termination signals and results in the RNA polymerase reading DNA past the normal (rho) transcription termination site. This process is called antitermination. These delayed early genes can lead directly to a lytic infection rather that a lysogenic infection.

Deciding whether to be in the lysogenic or lytic infection is based a variety of factors resulting from the current state of the host cell and the growth conditions present in the medium. If the integration process dominates upon infection, then the infection will be lysogenic. If the antitermination process dominates and the late genes end up being expressed, then the infection will be lytic. A prophage can be activated if the cell's growth conditions change - this results in a lysogenic infection becoming a lytic infection.

Biology 4: Genes and Society (1997, 1998)