HORT 250 - Biotechnology in Agriculture

Lecture 5 - Regulation of gene expression

In the last lecture I talked about how the information encoded in genes is read by an organism to produce proteins.

• RNA is transcribed from part of the DNA by RNA polymerase. The sequence of bases in DNA is copied in the RNA.
• Ribosomes translate the information in the RNA, the sequence of bases, into a sequence of amino acids in a protein.
• the ribosome identifies the first AUG triplet in the RNA and starts the protein with a methionine.
• the ribosome reads the RNA in a sequence of triplets or "codons" (3 bases at a time) with each triplet encoding a specific amino acid.
• translation of the RNA stops when a specific sequence of 3 bases (UAA, UAG or UGA) is read.

All of these elements of gene structure and expression are encoded in the DNA. In addition, the gene must also encode information that allows the appropriate and necessary regulation of this gene. Why is regulating the expression of genes an important function?

Consider the life of a simple bacterium that lives and grows in a solution of sucrose, perhaps this bacterium is enjoying life in the dregs left in the bottom of that forgotten Coke can under your bed. This bacterium makes proteins that allow it metabolise sucrose for energy and to make the carbon-containing compounds that it needs to grow and live. In the course of cleaning up your room (perhaps someone you are trying to impress is coming over for dinner, or maybe your parents are visiting) you find the Coke can and unceremoniously dump the contents into the jug of sour milk that was hiding at the back of the refrigerator. The bacteria that were quite content to be using sucrose to grow are thrown into a new environment where the only sugar that is available comes from the milk in the form of lactose. What are the bacteria going to do? I can propose three possible scenarios:

1. The bacteria are unable to alter their metabolism to use lactose, and so they die.
2. Regardless of the environment in which they live, the bacteria always make all of the proteins necessary to metabolise a wide range of food sources, including sucrose and lactose, so they can grow in the lactose solution.
3. The bacteria are able to activate genes and synthesise new proteins that are required to metabolise lactose, and so they adapt to the new environmental conditions.

From the standpoint of efficiency, which of these strategies makes the most sense? The ability to activate only those genes that are required for the bugs to grow in a specific set of circumstances is a more suitable survival strategy. This is similar to the idea of "just-in-time" deliveries in manufacturing. Factories have moved form maintaining large inventories of supplies that are stored in a warehouse to having supplies delivered as they are needed, just in time so that production on the line is not interrupted. Why have manufacturers made this switch? They found that maintaining large inventories was very expensive and that production could be run more efficiently if they only received their supplies as they were needed. If manufacturers had studied how bacteria organize and regulate their metabolism, this would have been obvious.

Bacteria are able to activate and repress specific genes in response to changes in their environment, such as the source of food. All organisms, from the simplest to the most complex, use a variety of methods to regulate the expression of their genomes. Here are a few more examples demonstrating the regulation of gene expression.

• When a virus infects a cell, it first activates a set of "early genes" in the viral genome that modify cellular metabolism to favor growth of the virus. Subsequently a set of "late genes" are switched on to replicate the virus and produce more proteins for the coat, the shell of the virus.
• Different organs in a multicellular organism express different sets of genes. The hormone responsible for regulating sugar metabolism, insulin, is produced by specific cells in the pancreas. But these cells do not make keratin, the protein in hair. In contrast, hair cells make keratin but do not make insulin. The genes that encode these proteins are present in the cells of both organs but they are expressed only in specific organs.

What would happen in a multicellular organism if there was no difference in gene expression between the cells and organs of that organism, i.e. all cells made exactly the same group of proteins. There would be no difference between those cells, they would all be alike. Therefore, there would be no differentiation, the organism would simply be a mass of identical cells - a blob! Clearly this is not the case. The regulated expression of genes is critical in establishing the properties of every organism, its form and function.

During development and differentiation of an organism from a fertilized egg, sets of genes are activated and repressed. The expression of these genes is regulated in time and space. As an example, I will use the development of a maize seed. The various stages of embryo development are outlined below.

• Fertilization - in plants, a double fertilization of both the egg and endosperm.
• Cell division and differentiation - establishing the root and shoot, and growth of the endosperm.
• Maturation - synthesis of storage reserves (proteins, oils and carbohydrates) to sustain the seedling after germination.
• Desiccation - loss of water resulting in the dormant seed.
• Germination - under favorable conditions of water, temperature, etc., leading to vegetative growth.

Each of these stages of development is accompanied by the expression of specific sets of genes. Some are expressed throughout the process of seed development, while others are expressed only at specific stages. There is also spatial control of gene expression. Genes that are responsible for making storage reserves are expressed in the endosperm and not in the embryo (root/shoot system). Specifc genes are required to make the shoot, another set for the root.

How do we know that specific genes are required, and different sets of proteins are made during these various stages of seed development?

• We can extract proteins from the seed as it develops and use a variety of methods to examine these proteins (e.g. separating them on a gel) and show there are striking differences depending on the stage of development.
• Geneticists have found mutations in specific genes that are involved in only one aspect of seed development. For example, one of the mutations that Gregor Mendel studied produced wrinkled pea seeds. These peas had a defect in a gene that makes some of the carbohydrate reserves that are stored in the seed. Because of this mutation, the seeds do not fill as well as normal wild type seeds and have a shrivelled, wrinkled appearance. But apart from this defect, the seed are perfectly viable and the rest of the plant is quite normal. So only one part of seed development has been affected by this gene that is specifically required for making seed storage reserves.
• Another mutation in maize affects the ability of seed to become dormant. Seed that do not make one specific protein are unable to become dormant, and so will sprout and germinate on the ear, before they are harvested. All other aspects of seed development are normal, but the seed cannot become dormant because they fail to express one gene at the correct time during development.

These are examples to emphasize the point that regulating the expression of genes is an essential part of life.

There are many ways in which gene expression can be regulated. Consider what happens if any of the following are changed while all the other steps are held constant, not changed.

• Gene copy number. What happens if you increase the number of copies of a gene from 1 to 10? If everything else remains the same, you would get ten times the amount of protein made.
• Gene transcription. In the most extreme case, transcription can be either off or on. But changes in the rate of transcription can also affect gene expression.
• RNA stability. If an RNA molecule is selectively degraded within the cell, then it is unavailable for translation. If the RNA is degraded before it is translated, then no protein is produced.
• Translation. If the RNA is present but is not translated, no protein is made. Alternatively, an RNA can be preferentially translated leading to production of more protein.
• Protein Function. A protein may require modification for activity, may be unstable, etc. These processes can also affect gene expression.

We can think of gene regulation in terms of trying to maintain the water in a bathtub at a particular level. When you want to fill the tub, you open the tap, similar to activating gene transcription. And when there is enough water in the tub, you switch off the tap; transcription is terminated when there is enough of that protein present. If the water utility company has either increased or decreased the water pressure in the system, the water will come out faster or slower. This is analagous to the number of genes for that protein - more genes, more RNA. But remember there are a number of other ways to regulate the water level in the tub, and we can compare these to the various steps after transcription. You can let the water out via the drain, or, if you have a vigorous 4-year old, he can remove water in some less conventional and more annoying (at least from a parental viewpoint) ways!

The most important and widely used method to regulate gene expression is to modulate transcription. This makes good sense because it is the first step in the pathway.

The part of the gene that carries the information about where and when a gene should be expressed is known as the promoter. The promoter normally lies upstream of the site for starting transcription. The sequence of bases in the DNA of the promoter directs the expression of that gene.

DNA by itself cannot transcribe a molecule of RNA. So the promoter works by interacting with other molecules, typically proteins. These proteins must include the RNA polymerase that transcribes RNA as well as other regulators that modulate transcription.

We will first examine how bacteria regulate the expression of specific genes.

E. coli is able to use lactose as a source of carbon for growth, b -galactosidase converts lactose into glucose. The gene encoding b -galactosidase is regulated in response to growth conditions. When lactose is not present in the growth medium, this enzyme is not produced, but is synthesized when lactose is added. How does the bacterium regulate its metabolism in this manner? A more detailed description of this regulatory mechanism is available from the MIT Biology Hypertextbook web site, which includes a chapter on regulation of lactose metabolism.

The bacterium produces a protein known as a repressor. This repressor is made all the time, regardless of growth conditions. When there is no lactose in the medium, this repressor binds to a specific region in the promoter of the gene encoding b -galactosidase. By binding to the DNA at this specific site, the repressor prevents RNA polymerase from binding and transcribing this gene. The gene is inactive when lactose is present in the medium. The repressor not only binds to DNA, but it binds to a highly specific bit of DNA, in the promoter of this gene. The repressor protein is able to recognise a specific sequence of bases in the DNA and bind only there. It will not bind to just any piece of DNA. This specificity is an essential component of regulation.

When lactose is present, the bacterium needs to activate the gene for b-galactosidase to utilize this food source. The repressor sitting on the promoter also has a site for binding lactose. When lactose binds to the repressor it changes the shape of the protein so that it no longer binds to the promoter. When the repressor is not blocking the promoter, RNA polymerase can bind, transcribe the gene and produce b -galactosidase.

What happens when b-galactosidase has converted all the lactose to glucose? There is no longer any lactose to bind the repressor, so the repressor can once again bind to the promoter and switch off expression of this gene.

A second example of regulation in E. coli is the genes for biosynthesis of the amino acid tryptophan. Tryptophan is one of the essential amino acids required to make proteins, as well as a number of other compounds. The bacterium can sometimes obtain tryptophan from its environment, for example if the bacterium is growing in a medium containing trytophan. But when there is no available source of tryptophan, the bacterium has to make its own supply of this amino acid. Under these conditions, the genes encoding enzymes to make tryptophan are active. But if tryptophan is present, these genes should be switched off. This is the opposite scenario to that described for lactose.

Again, the bacterium produces a repressor protein. When there is no tryptophan around, the repressor cannot bind to the promoter of the tryptophan synthesis gene. However, when there is excess tryptophan in the cell, some of this binds to the repressor, alters its shape and it can then bind to the promoter. When the repressor is in place, RNA polymerase cannot bind to the promoter and transcription does not occur.

When the supply of tryptophan is depleted, there is no longer any of the amino acid to bind to the repressor. When the repressor is not bound to the promoter expression of the tryptophan synthesis genes is activated. There is a similar explanation of this mechanism in a chapter of the MIT Biology Hypertextbook.

The critical points to remember from this lecture are the following:

• regulating the expression of genes, the information carried in the genome, is essential for all organisms.
• transcription of DNA into RNA is the most widely used mechanism to regulate expression of genes, although there are many other mechanisms.
• the promoter is the part of the gene that plays a major role in this regulation.
• promoters direct RNA polymerase to bind to the gene and initiate transcription.
• other molecules, usually proteins, bind to specific DNA sequences in the promoter and can modulate expression of individual genes.

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