Chapter 18 - Control of Gene Expression in Bacteria
Chapter 18 - Control of Gene Expression in Bacteria BIOL 2311
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Date Created: 07/27/16
Chapter 18 – Control of Gene Expression in Bacteria MingHan Lu Cells are extremely selective about which genes are expressed, in what amounts, and when. Gene expression is the process of converting information that is archived in DNA into molecules that actually do things in the cell. o It occurs when a protein or other gene product is synthesized and active. 18.1 An Overview of Gene Regulation and Information Flow Each sugar should induce a different response from the E. coli cells in your intestine. Cells need to regulate which proteins they produce. Mechanisms of Regulation The flow of information from DNA to activation of the final gene product occurs in three steps, represented by arrows in the following diagram. DNA mRNA protein activated protein Gene expression can be controlled at any of these steps. The arrow from DNA to RNA represents transcription—the making of messenger RNA (mRNA). The arrow from RNA to protein represents translation, in which ribosomes read the information in mRNA and use that information to synthesize a protein. The arrow from protein to activated protein represents posttranslational modifications that can lead to changes in shape and activity. 3 mechanisms to regulate flow of information from DNA protein: 1. The Cell would avoid making the mRNAs for particular enzymes. If there is no mRNA, then ribosomes cannot make the gene product. Transcriptional control occurs when regulatory proteins affect RNA polymerase’s ability to bind to a promoter and initiate transcription. 2. If the mRNA for an enzyme has been made, the cell could prevent the mRNA from being translated into protein. Translational control occurs when regulatory molecules alter the length of time an mRNA survives, or affect translation initiation or elongation. 3. Many proteins have to be activated by chemical modification, such as the addition of a phosphate group. Regulating this final step is posttranslational control. Transcriptional control is particularly important due to its efficiency—it saves the most energy for the cell, because it stops the process of gene expression at the earliest possible point. Translational control allows a cell to make rapid changes in the amounts of different proteins because the mRNA is already present and available for translation. Posttranslational control provides the most rapid response of all three mechanisms because only one step is needed to activate an existing protein. Among these mechanisms of gene regulation, there is a clear tradeoff between the speed of response and the conservation of ATP, amino acids, and other resources. Transcriptional control is slow but efficient in resource use Posttranslational control is fast but energetically expensive. Chapter 18 – Control of Gene Expression in Bacteria MingHan Lu Some genes—such as those that code for the enzymes required for glycolysis—are transcribed all the time, or constitutively. Gene expression is not an allornone proposition. Genes are not just “on” or “off”— instead, the level of expression can vary between these extremes. The ability to regulate gene expression allows cells to respond to changes in their environment. Metabolizing Lactose – A Model System In early studies of gene regulation, a key model system was the metabolism of the sugar lactose in E. coli. E. Coli can use a wide variety of sugars for ATP production, via cellular respiration or fermentation. o These sugars also serve as raw material in the synthesis of amino acids, vitamins and other complex compounds. o Glucose, however, is E. coli’s preferred carbon source—meaning that it is the source of energy and carbon atoms that the organism uses most efficiently. Lactose, the sugar found in milk, can also be used by E. coli, but it is not used until glucose supplies are depleted. o Lactose is a disaccharide made up of one molecule of glucose and one molecule of galactose. To use lactose, E. coli must first transport the sugar into the cell. o Once lactose is inside the cell, the enzyme galactosidase catalyzes a reaction that breaks down the disaccharide into glucose and galactose. The glucose released by this reaction goes directly into the glycolytic pathway; other enzymes convert the galactose to a substance that can also be processed in the glycolytic pathway. o E. coli produces high levels of galactosidase only when lactose is present in the environment. o Lactose itself regulates the gene for galactosidase – meaning that the lactose acts as an inducer. An inducer is a small molecule that triggers transcription of a specific gene. 18.2 Identifying Regulated Genes To understand how E. coli controls production of galactosidase and the transport protein that brings lactose into the cell, Jacob and Monod isolated and analyzed mutants. o Their goal was to find E.coli cells that could not metabolize lactose. Cells that can’t use lactose must lack either galactosidase or the lactose transporter protein. To find mutants that are associated with a particular trait, a researcher has to complete two steps: 1. Generate a large number of individuals with mutations at random locations in their genomes. Chapter 18 – Control of Gene Expression in Bacteria MingHan Lu a. Monod accomplished this step by exposing E. coli populations to mutagens—X rays, UV light, or chemicals that damage DNA and increase mutation rates. 2. Screen the treated individuals for mutants with defects in the process or biochemical pathway in question—this case case, defects in lactose metabolism. Several Genes Are Involved in Lactose Metabolism Three types of mutants: 1. In one class, the mutant cells were unable to cleave lactose – even when lactose was in the medium and transported into cells to induce production of the galactosidase. Jacob and Monod concluded that these mutants must lack a functioning version of the galactosidase protein and, therefore, the gene that encodes galactosidase is defective. This gene was designated lacZ, and the mutant allele lacZ. 2. Cells cannot accumulate lactose. Jacob and Monod hypothesized that the mutant cells had defective copies of the membrane protein responsible for transporting actose into the cell. This protein was identified and named galactoside permease. Gene is called lacY. The mutant allele is lacY. 3. Cells cleave lactose even if lactose is absent as an inducer. These mutants made the proteins all the time—even if no lactose was present. Cells that are abnormal because they produce a product at all times are called constitutive mutants. The gene that was mutated to produce constitutive galactosidase and galactoside permease expression was named lacI. The letter I signified that these mutants did not need an inducer—lactose—to express galactosidase or galactoside permease. lacI mutations have a defect in gene regulation because gene expression occurs with or without lactose. Jacob and Monod had succeeded in identifying three genes involved in lactose metabolism: lazZ, lacY, and lacI. They concluded that lacZ and lac Y code for proteins required for the metabolism and import of lactose, while lacI is responsible for some sort of regulatory function. o When lactose is absent, the lacI gene or gene product shuts down the expression of lacZ and lacY. But when lactose is present, the opposite occurs—transcription of lacZ and lacY is induced. 18.3 Negative Control of Transcription There are two ways that transcription can be regulated: by negative control or positive control. 1. Negative control occurs when a regulatory protein called a repressor binds to DNA and shuts down transcription. 2. Positive control occurs when a regulatory protein called an activator binds to DNA and triggers transcription. The IacI gene produces a repressor protein that exerts negative control over lacZ and lacY gene gene transcription. Chapter 18 – Control of Gene Expression in Bacteria MingHan Lu The repressor was proposed to bind directly to DNA at or near the promoter for the lacZ and lacY genes. Lactose interacts with the repressor in a way that makes the repressor release from its binding site. In negative control, the repressor is the parking brake; lactose releases the brake. Lactose removes the suppressor. The lacI gene codes for a repressor protein that exerts negative control on lacZ and lacY. o Lactose acts as an inducer by causing the repressor to release from DNA and ending negative control. The Operon Model Key conclusion was that the genes for galactosidase and galactoside permease are controlled together and transcribed into a single mRNA. o Coined the term operon for a set of coordinately regulated bacterial genes that are transcribed together into one mRNA. The group of genes involved in lactose metabolism was termed the lac operon. A gene called lacA was found to be adjacent to lacY and lacZ and transcribed as part of the same operon. The lacA gene codes for the enzyme transacetylase. o This enzyme catalyzes reactions that allow certain types of sugars to be exported from the cell when they are too abundant and could harm the cell. 3 hypotheses are central to the JacobMonod model of lac operon regulation: 1. The lacZ, lacY, and lacA genes are adjacent and are transcribed into one mRNA intiated from the single promoter of the lac operon. This is known as cotranscription, and it results in the coordinated expression of the three genes. 2. The repressor is a protein encoded by lacI that binds to DNA and prevents transcription of the lac operon genes (lacZ, lacY, and lacA). Jacob and Monod proposed that lacI is expressed constitutively, and that the repressor binds to a section of DNA in the lac operon called the operator. 3. The inducer(lactose) binds to the repressor. When it does, the repressor changes shape. The shape change causes the repressor to come off the DNA. a. This form of control over protein function is allosteric regulation. In allosteric regulation, a small molecule binds to a protein and causes it to change its shape and activity. When the inducer binds to the repressor, the repressor can no longer bind to DNA and transcription can proceed. How Does Glucose Regulate the lac Operon? Transcription of the lac operon is drastically reduced when glucose is present in the environment—even when lactose is available to induce galactosidase expression. o This makes sense, given that glucose is E. coli’s preferred carbon source. When glucose is already present, the cell doesn’t need to cleave lactose as a way of acquiring glucose. Chapter 18 – Control of Gene Expression in Bacteria MingHan Lu Glucose inhibits the lactose transport activity of galactoside permease through a chain of molecular events. When both glucose and lactose are present in the environment, the transport of lactose into the cell is inhibited. o Because lactose does not accumulate in the cytoplasm, the repressor remains bound to the operator. o In contrast, when glucose levels outside the cell are low, galactoside permease is active. If lactose is present, it is transported into the cell and induces lac operon expression. The mechanism of glucose preventing the transport of inducer is known as inducer exclusion. o Inducer exclusion affects the activity of many different sugar transporters in addition to galactoside permease. It allows E. coli to preferentially use glucose, even when other sugars also present outside the cell. Why Has the lac Operon Model Been So Important? Many bacterial genes and operons are under negative control by repressor proteins. Gene expression is regulated by physical contact between regulatory proteins and specific regulatory sites in DNA. o When a rapid change in lac operon activity is needed, it does not require changes in the transcription or translation of new repressor proteins. Instead, the activity of existing repressor proteins is altered. Posttranslational control is best when a rapid response is needed. 18.4 Positive Control of Transcription Positive control is an important way of controlling transcription. In positive control, an activator protein binds to a regulatory sequence in DNA when genes are turned on. o When bound to DNA, the activator interacts with RNA polymerase to increase the rate of initiating transcription. The ara operon provides an important example of positive control and of the process of science. This operon contains contains 3 genes that allow E. coli to use the sugar arabinose. o Without arabinose in the environment, the ara operon is not transcribed. o But when abarinose is present, transcription of the ara operon is turned on by an activator protein called AraC. The ara operon and an adjacent gene, araC, that codes for the araC activator. AraC protein is allosterically regulated by arabinose. When bound to arabinose, two copies of the AraC protein attach to a regulatory sequence of DNA called the ara initiator that lies just upstream of the promoter. o This interaction between AraC and the RNA polymerase helps to dock the polymerase to the promoter and accelerate the initiation of transcription. o AraC is both an activator and a repressor. In the absence of arabinose, the two copies of AraC protein remain together; but while one araC copy remains bound Chapter 18 – Control of Gene Expression in Bacteria MingHan Lu to the initiator, the other copy now bind to a different regulatory site in DNA, the ara operator. 18.5 Global Gene Regulation Global gene regulation is the coordinated regulation of many genes. o It is needed for responses that require the expression of dozens or even hundreds of genes. o There are other means of global gene regulation, such as grouping genes into a regulon—a set of separate genes or operons that contain the same regulatory sequences that are controlled by a single type of regulatory protein. Regulon consists of many genes that are scattered across the genome. o All of these genes are controlled are controlled by the same type of repressor protein that binds to the same operator sequences near the promoter of each gene. o When an environmental change triggers the removal of the repressor protein from all the operators, every gene in the regulon is transcribed. o Regulons can be under negative control by a repressor protein or positive control by an activator protein. Interactions among protein regulators and the DNA sequences they bind produce finely tuned control over gene expression, regulating individual genes, operons, or large sets of genes.
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