Gene regulation in bacteria

SIGNIFICANCE:Gene regulation is the process by which the synthesis of gene products is controlled. The study of gene regulation in bacteria has led to an understanding of how cells respond to their external and internal environments.

The Discovery of Gene Regulation

In 1961, a landmark paper by French researchers François Jacob and Jacques Monod outlined what was known about genes involved in the breakdown of sugars, the synthesis of amino acids, and the reproduction of a bacterial virus called lambda phage (λ phage). Jacob and Monod described in detail the of enzymes that break down the sugar lactose. These enzymes were induced by adding the sugar or, in some cases, structurally related molecules to the media. If these molecules were removed, the enzymes altering lactose were no longer synthesized. Bacteria without the lacI gene (lacI^-) produced the enzymes for metabolizing lactose whether or not the inducer was present.

Although bacteria normally have only one copy of each gene locus, they can be given extra copies of selected genes by transforming them with a plasmid containing the genes of interest. Thus, bacteria that are at a locus can be produced. When Jacob and Monod produced bacteria heterozygous for the lacI gene (lacI- / lacI+), they functioned like normal bacteria (lacI+), indicating that thelacI+ allele was dominant to the lacI^- allele. Certain alleles of the operator site, lacOC, result in the synthesis of lactose-altering enzymes whether or not the inducer was present and even when lacI+ was present. These observations suggested that the lacI+ gene specified a repressor that might bind to lacO+and block transcription of the genes involved in lactose metabolism. Jacob and Monod concluded that inducers interfered with the repressor’s ability to bind to lacO+. This allowed transcription and translation of the lactose operon. In their model, the protein is unable to bind to the altered operator site, lacOC. This explained how certain mutations in the operator caused the enzymes for lactose to be continuously expressed.

Seeing a similarity between the expression of the genes for lactose metabolism, the genes for synthesis, and the genes for lambda phage proliferation, Jacob and Monod proposed that all genes might be under the control of operator sites that are bound by repressor proteins. An consists of the genes that the operator controls. Although the vast majority of operons have operators and are regulated by a repressor, there are some operons without operator sites that are not controlled by a repressor. Generally, these operons are regulated by an inefficient or by transposition of the promoter site, whereas some are inhibited by attenuation, a more complex interaction occurring during transcription and translation. The only controlling site absolutely necessary for is the promoter site, where binds.

Lactose Operon: Negatively Controlled Genes

The lactose operon (lacZYA) consists of three controlling sites (lacCRP, lacPZYA, and lacO) and three structural genes (lacZ, lacY, and lacA). The lactose operon is controlled by a neighboring operon, the lactose regulatory operon, consisting of a single controlling site (lacPI) and a single structural gene (lacI). The order of the controlling sites and structural genes in the bacterial chromosome is lacPI, lacI, lacCRP, lacPZYA, lacO, lacZ, lacY, lacA. Transcription of the regulatory operon proceeds to the right from the promoter site, lacPI. Similarly, transcription of the L-arabinose operon occurs rightward from lacPZYA. A cyclic-adenosine monophosphate receptor (CRP) bound by cyclic-adenosine monophosphate (cAMP), referred to as a CRP-cAMP complex, attaches to the lacCRP site.

The lacI gene specifies the protein subunit of the lactose repressor, a tetrameric protein that binds to the operator site, lacO, and blocks transcription of the operon. The lacZ gene codes for beta-galactosidase, the that cleaves lactose into galactose plus glucose. This enzyme also converts lactose into the effector molecule allolactose, which actually binds to the repressor inactivating it. The lacY gene specifies the enzyme, known as the “lactose permease,” that transports lactose across the plasma membrane and concentrates it within the cell. The lacA gene codes for an enzyme called transacetylase, which adds acetyl groups to lactose.

In the absence of lactose, the repressor occasionally diffuses from the operator, allowing RNA polymerase to attach to lacPZYA and make a single RNA transcript. This results in extremely low levels of enzymes called the “basal” level. With the addition of lactose, a small amount of allolactose binding to the repressor induces a conformational change in the repressor so that it no longer binds to lacO. The levels of permease and beta-galactosidase quickly increase, and within an hour the enzyme levels may be one thousand times greater than they were before lactose was added.

Normally, cells do not produce levels of lactose or enzymes that are more than one thousand times greater than basal level because the lactose operon is regulated by catabolite repression. As cells synthesize cellular material at a high rate, lactose entrance and synthesis are inhibited, whereas cAMP secretion into the environment is increased. This causes most of the CRP-cAMP complex to become CRP. CRP is unable to bind to lacCRP and promote transcription from lacPBAD.

If lactose is removed from the fully induced operon, the repressor quickly binds again to lacO and blocks transcription. Within a few hours, lactose and proteins return to their basal levels. Since the lactose operon is induced and negatively regulated by a repressor protein, the operon is classified as an inducible, negatively controlled operon.

Arabinose Operon: Positively Controlled Genes

The L-arabinose operon (araBAD) has been extensively characterized since the early 1960s by American researchers Ellis Englesberg, Nancy Lee, and Robert Schleif. This operon is under the control of a linked regulatory operon consisting of (araC, araO2) and (araPC, araO1). The parentheses indicate that the regions overlap: araO2 is an operator site in the middle of araC, whereas araP_C and araO1 represent a promoter site and an operator site respectively, which overlap. The order of the controlling sites and genes for the regulatory operon and the L-arabinose operon is as follows: (araC, araO2) (araPC, araO1), araCRP, araI1, araI2, araPBAD, araPB, araA, araD. RNA polymerase binding to araPC transcribes araC leftward, whereas RNA polymerase binding to araPBAD transcribes araBAD rightward.

The araA gene specifies an isomerase that converts L-arabinose to L-ribulose, the araB gene codes for a that changes L-ribulose to L-ribulose-5-phosphate, and the araD gene contains the information for an epimerase that turns L-ribulose-5-phosphate into D-xylulose-5-phosphate. Further metabolism of D-xylulose-5-phosphate is carried out by enzymes specified by genes in other operons.

The araC product is in equilibrium between two conformations, one having repressor activity and the other having activity. The conformation that functions as an activator is stabilized by the binding of L-arabinose or by certain mutations (araCC). In the absence of L-arabinose, almost all the araC product is in the repressor conformation; however, in the presence of L-arabinose, nearly all the araC product is in the activator conformation.

In the absence of L-arabinose, bacteria will synthesize only basal levels of the lactose regulatory protein and the enzymes involved in the breakdown of L-arabinose. The repressor binding to araO2 prevents araC transcription beginning at araPC from being completed, whereas the repressor binding to araI1 prevents araBAD transcription beginning at araPBAD.

The addition of L-arabinose causes the repressor to be converted into an activator. The activator binds to araI1 and araI2 and stimulates araBAD transcription. An activator is absolutely required for the metabolism of L-arabinose since bacterial cells with a defective or missing L-arabinose regulatory protein, araC^-, only produce basal levels of the L-arabinose enzymes. This is in contrast to what happens to the lactose enzymes when there is a missing lactose regulatory protein, lacI^-. Because of the absolute requirement for an activator, the L-arabinose operon is considered an example of a positively controlled, inducible operon.

Transcription of the araBAD operon is also dependent upon the cyclic-adenosine monophosphate receptor protein (CRP), which exists in two conformations. When excessive and cellular constituents are being synthesized from L-arabinose, cAMP levels drop very low in the cell. This results in CRP-cAMP acquiring the CRP conformation and dissociating from araCRP. When this occurs, the araBAD operon is no longer transcribed. The L-arabinose operon is controlled by very much like the lactose operon.

Tryptophan Operon: Genes Controlled by Attenuation

The tryptophan operon (trpLEDCBA) consists of the controlling sites and the genes that are involved in the synthesis of the amino acid tryptophan. The order of the controlling sites and genes in the tryptophan operon is as follows: (trpP, trpO), trpL, trpE, trpD, trpC, trpB, trpA. RNA polymerase binds to trpP and initiates transcription at the beginning of trpL.

An inactive protein is specified by an unlinked regulatory gene (trpR). The regulatory protein is in equilibrium between its inactive and its repressor conformation, which is stabilized by tryptophan. Thus, if there is a high concentration of tryptophan, the repressor binds to trpO and shuts off the tryptophan operon. This operon is an example of an operon that is repressible and negatively regulated.

The tryptophan operon is also controlled by a process called attenuation, which involves the mRNA transcribed from the leader region, trpL. The significance of leader region mRNA is that it hydrogen-bonds with itself to form a number of hairpinlike structures. Hairpin-III interacts with the RNA polymerase, causing it to fall off the DNA. Any one of several hairpins can form, depending upon the level of tryptophan in the environment and the cell. When there is no tryptophan in the environment, the operon is fully expressed so that tryptophan is synthesized. This is accomplished by translation of the leader region right behind the RNA polymerase up to a couple of tryptophan codons, where the ribosomes stall. The stalled ribosomes cover the beginning of the leader mRNA in such a way that only hairpin-II forms. This hairpin does not interfere with transcription of the rest of the operon and so the entire operon is transcribed.

When there is too much tryptophan, the operon is turned off to prevent further synthesis of tryptophan. This is accomplished by translation of the leader region up to the end of the leader peptide. Ribosomes synthesizing the leader peptide cover the leader mRNA in such a way that only hairpin-III forms. This hairpin causes attenuation of transcription.

In some cases, the lack of amino acids other than tryptophan can result in attenuation of the tryptophan operon. In fact, cells starved for the first four amino acids (N-formylmethionine, lysine, alanine, and isoleucine) of the leader peptide result in attenuation. When these amino acids are missing, hairpins-I and -III both form, resulting in attenuation because of hairpin-III.

Flagellin Operons: Operons Controlled by Transposition

Some pathogenic bacteria change their flagella to avoid being recognized and destroyed by the host’s immune system. This change in flagella occurs by switching to the synthesis of another flagellar protein. The phenomenon is known as phase variation. The genes for flagellin are in different operons. The first operon consists of a promoter site, an operator site, and the structural gene for the first flagellin (flgPH1, flgO1, flgH1). The first operon is under the negative control of a repressor specified by the second operon. The second operon also specifies the second flagellin and a transposase that causes part of the second operon to reverse itself. This portion of the operon that “flips” is called a “transposon.” The promoter sites for the transposase gene (flgT2), flagellin gene (flgH2), and repressor gene (flgR2) are located on either side of the transposase gene in sequences called inverted repeats. Transcription from both promoters in the second operon occurs from left to right: flgPT2, flgT2, flgPH2R2, flgH2, flgR2.

When the second operon is active, the repressor binds to flgO1, blocking the synthesis of the first flagellin (flgH1). Consequently, all bacterial flagella will be made of the second flagellin (flgH2). Occasionally, the transposase will catalyze a event between the inverted repeats, which leads to the being reversed. When this occurs, neither flgH2 nor flgR2 is transcribed. Consequently, the first operon is no longer repressed by flgR2, and flgH1 is synthesized. All the new flagella will consist of flgH1 rather than flgH2.

Impact and Applications

Many of the genetic procedures developed to study gene regulation in bacteria have contributed to the development of genetic engineering and the production of biosynthetic consumer goods. One of the first products to be manufactured in bacteria was human insulin. The genes for the two insulin subunits were spliced to the lactose operon in different populations of bacteria. When induced, each population produced one of the subunits. The cells were cracked open, and the subunits were purified and mixed together to produce functional human insulin. Many other products have been made in bacteria, yeast, and even plants and animals.

Considerable progress has been made toward introducing genes into plants and animals to change them permanently. In most cases, this is difficult to do because the controlling sites and gene regulation are much more complicated in higher organisms than in bacteria. Nevertheless, many different species of plants have been altered to make them resistant to desiccation, herbicides, insects, and various plant pathogens. Although curing genetic defects by introducing good genes into animals and humans has not been very successful, animals have been transformed so that they produce a number of medically important proteins in their milk. Goats have been genetically engineered to release tissue plasminogen activator, a valuable enzyme used in the treatment of heart attack and stroke victims, into their milk. Similarly, sheep have been engineered to secrete human alpha-1 antitrypsin, useful in treating emphysema. Cattle that produce more than ten times the milk that sheep or goats produce may potentially function as factories for the synthesis of all types of valuable proteins specified by artfully regulated genes.

Key Terms

• allelean alternative form of a gene; for example, lacI+, lacI-, andlacIS are alleles of the lacI gene
• controlling sitea sequence of base pairs to which regulatory proteins bind to affect the expression of neighboring genes
• genea sequence of base pairs that specifies a product (either RNA or protein); the average gene in bacteria is one thousand base pairs long
• operonone or more genes plus one or more controlling sites that regulate the expression of the genes
• transcriptionthe use of DNA as the template in the synthesis of RNA
• translationthe use of an RNA molecule as the guide in the synthesis of a protein

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