Inducible Genes and Repressible Genes
In order for genetic information stored in the form of DNA sequence to be translated, the information must first be transcribed into messenger RNA (mRNA); mRNA is synthesized by an enzyme, RNA polymerase, which uses the DNA sequences as a template for making a single strand of RNA that can be translated into proteins. The proteins are the functional gene products that act as enzymes or structural elements for the cell. In many cases the RNA by itself or after modification can also act as enzyme or form structural elements. The process by which DNA is transcribed and then translated is referred to as “gene expression.”
Some genes are always expressed in bacterial cells; that is, they are continually being transcribed into mRNA, which is translated into functional proteins (gene products) of the cell. The genes involved in using glucose as an energy source are included in this group. Other genes are inducible (expressed only under certain specific conditions). The genes for using lactose as an energy source are included in this group. The lactose operon is made up of three structural genes: LacZ, LacY, and LacA, which encode for beta-galactosidase, lac permease, and a transacetylase, respectively. The beta-galactosidase is the enzyme that converts lactose into glucose and galactose. Lac permease is a transmembrane protein that is necessary for lactose uptake, and transacetylase transfers the acetyl group from coenzyme A to beta galactosides. However, only beta-galactosidase and lac permease play an active role in the regulation of
lac operon. Another regulatory gene, lacI, which codes for the lac repressor, is not included in this operon but lies nearby and is always expressed.
As early as the 1940’s François Jacob, Jacques Monod, and their associates were studying the mechanisms by which beta-galactosidase was induced in Escherichia coli. They discovered that in the absence of lactose in a cell, the repressor protein binds at the operator sequence of the lac operon. Under these conditions, transcription of genes in the operon is inhibited since the RNA polymerase is physically prevented from binding to the promoter when the repressor is already bound. This occurs because the promoter and operator sequences are overlapping. The lactose (lac) operon is, therefore, under
negative control. When lactose is present, an altered form of the lactose known as allolactose attaches to the repressor in such a way that the repressor can no longer bind to the operator. With the operator sequence vacant, it is possible for the RNA polymerase to bind to the promoter and to begin transcription of the operon genes. Lactose (or its metabolite) serves as an inducer for transcription. Only when it is present are the lactose operon genes transcribed. The lactose operon is, therefore, an inducible operon. In 1965, Jacob and Monod were awarded the Nobel Prize in Physiology or Medicine in recognition of their discoveries concerning the genetic control of enzyme synthesis.
Lac Operon Expression in the Presence of Glucose
When a culture of E. coli is given equal amounts of glucose and lactose for growth and is compared with cultures given either glucose alone or lactose alone, the cells given two sugars do not grow twice as fast, but rather show two distinct growth cycles. Beta-galactosidase is not synthesized initially; therefore, lactose is not used until all the glucose has been metabolized. Laboratory observations show that the presence of lactose is necessary but not a sufficient condition for the lactose (lac) operon to be expressed. An activator protein must bind at the promoter in order to unravel the DNA double helix so that the RNA polymerase can bind more efficiently. The activator protein binds only when there is little or no glucose in the cell. If glucose is available, it is preferred over other sugars because it is most easily metabolized to make energy in the form of adenosine triphosphate (ATP). ATP is made through a series of
reactions from an intermediate molecule, cyclic adenosine monophosphate (cAMP). The cAMP concentration decreases when ATP is being made but builds up when no ATP synthesis occurs. When the glucose has been used, the concentration of cAMP rises. The cAMP binds to a specific receptor protein to form the CAP complex. The CAP binds at a specific DNA site upstream from the lac promoter and increases the affinity of mRNA polymerase for the operon’s promoter. With the activator bound, transcription of the lac operon genes can occur. This regulatory mechanism is known as catabolite repression.
The activation of a DNA-binding protein by cAMP is a global control mechanism. The lactose operon is only one of many that are regulated in this way. Global control allows bacteria to prevent or turn on transcription of a group of genes in response to a single signal. It ensures that the bacteria always utilize the most efficient energy source if more than one is available. This type of global control only occurs, however, when the operon is also under the control of another DNA-binding protein (the lac repressor in the case of the lac operon), which makes the operon inducible or repressible or both. Control of transcription through the binding of an activator protein is an example of positive control, since binding of the activator turns on gene expression.
Impact and Applications
Jacob and Monod developed the concept of an operon as a functional unit of gene expression in bacteria. What they learned from studying the lac operon has led to a more general understanding of gene transcription and genetic regulatory pathways. The operon concept has proven to be a universal mechanism by which bacteria organize their genes. Although genes of higher cells (eukaryotes) are not usually organized in operons and although negative control of expression is rare in them, similar positive control mechanisms occur in both bacterial and eukaryotic cells. Studies of the lac operon have made possible the understanding of how DNA-binding proteins can attach to a promoter to enhance transcription.
The operon model defined by Jacob and Monod established that regulators of genetic information in addition to the structural gene itself affect protein synthesis. A single regulator gene could control the synthesis of several different proteins. Another significant idea was that the presence or absence of external agents can influence the synthesis of the proteins.
One of the important applications of the lac operon has been in the development of cloning vectors. The inducible promoter and an easily assayable structural gene, beta-galactosidase, are the two features that have been very useful in molecular and genetic studies. When the host strain carrying the beta-galactosidase vector is grown in the presence of the inducer IPTG and the chromogenic beta-galactosidase substrate X-gal, the colonies are blue. Blue-white screening has cleverly been used in many laboratories to identify the mutations.
The functionality of genes can also be assessed by creating lac fusions with promoter-less beta-galactosidase. The sensitive beta-galactosidase assay can then be performed to detect the expression of proteins from the target promoters. LacZ used in such a way is called reporter gene. Since the elucidation of the lac operon, the insights gained from these studies have been extensively used for negative inducible control of gene expression, to control expression in eukaryotic systems in vivo and to generate conditional gene silencing in complex systems.
Key terms
activator
:
a protein that binds to DNA to enhance a gene’s conversion into a product that can function within the cell
operator
:
a sequence of DNA adjacent to (and usually overlapping) the promoter of an operon; binding of a repressor to this DNA prevents transcription of the genes that are controlled by the operator
operon
:
a group of genes that all work together to carry out a single function for a cell
promoter
:
a sequence of DNA to which the gene expression enzyme (RNA polymerase) attaches to begin transcription of the genes of an operon
repressor
:
a protein that prevents a gene from being made into a functional product when it binds to the operator
Bibliography
Judson, Horace Freeland. The Eighth Day of Creation: Makers of the Revolution in Biology. Rev. ed. New York: Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 1996. A noted text that provides an interesting account of the personalities behind the discoveries that form the basis of modern molecular biology.
Liao, S., et al. “Transgenic LacZ Under Control of Hec-6st Regulatory Sequences Recapitulates Endogenous Gene Expression on High Endothelial Venules.” Proceedings of the National Academy of Sciences 13, no. 104 (March, 2007): 4577-4582. This article illustrates the application of LacZ for studying the endothelial venule gene which affects chronic inflammation in autoimmunity, graft rejection, and microbial infection.
Müller-Hill, Benno. The “Lac” Operon: A Short History of a Genetic Paradigm. New York: Walter de Gruyter, 1996. Using a unique combination of personal anecdotes and present-day science, describes the history and present knowledge of a paradigmatic system, the lac operon of Escherichia coli. Illustrated.
Ptashne, Mark, and Walter Gilbert. “Genetic Repressors.” Scientific American 222 (June, 1970). Summarizes repression mechanisms that turn genes on and off, using the lac operon and the lambda bacterial virus as models.
Santillan, M., and M. C. Mackey. “Quantitative Approaches to the Study of Bistability in the Lac Operon of Escherichia coli.” Journal of the Royal Society Interface 6, no. 5, supp. 1 (August, 2008): S29-39. This paper describes the significance and the history of the lac operon. In addition it describes the bistable behavior in detail and incorporating mathematical models.
Tijan, Robert. “Molecular Machines That Control Genes.” Scientific American 271 (February, 1995). Discusses regulatory proteins that direct transcription of DNA and what happens when they malfunction.
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