Microbial Genetics Lecture 5 PDF

The Libyan Academy, Department of Microbiology Dr. Mohamed Bumadian May, 2023  Lecture 5  Regulation of Bacterial Gene Expression Feedback Inhibition Repression and Induction (a) Lactose catabolism...

The Libyan Academy, Department of Microbiology Dr. Mohamed Bumadian May, 2023  Lecture 5  Regulation of Bacterial Gene Expression Feedback Inhibition Repression and Induction (a) Lactose catabolism (b) Synthesis of tryptophan (c) Positive regulation (glucosr, lactose)  Regulation of Bacterial Gene Expression Feedback Inhibition Stops enzymes that have already been synthesized. A cell's genetic machinery and its metabolic machinery are integrated and interdependent. Recall that the bacterial cell carries out an enormous number of metabolic reactions. The common feature of all metabolic reactions is that they are catalyzed by enzymes. Also recall that feedback inhibition stops a cell from performing unneeded chemical reactions. Feedback inhibition stops enzymes that have already been synthesized. ‫السمة المشتركة‬ ‫متكامل ومترابط‬  In nature, microbes rarely produce amino acids in excess of their own needs because feedback inhibition (end-product inhibition) prevents wasteful production of primary metabolites. This control mechanism stops the cell from making more of a substance than it needs and thereby wasting chemical resources. ‫يمنع اإلسراف في إنتاج‬ ‫وبالتالي إهدار الموارد الكيميائية‬  Substrate Pathway Operates Pathway Shuts Down Enzyme 1 Allosteric site Allosteric site Bound end Intermediate A -product Feedbacj Inhibition Enzyme 2 Intermediate B Enzyme 3 End- product Q What is meant by Feedback inhibition?  E+S→ES Complex→E+P The term holoenzyme refers to the active enzyme with its nonprotein component (a metal ion, cofactor or organic molecule, coenzyme). The term apoenzyme The protein component of an enzyme, to which the coenzyme attaches to form an active enzyme. If the nonprotein part is a metal ion such as Zn 2+ or Fe2+, it is called a cofactor. If it is a small organic molecule, it is termed a coenzyme such as NAD and FAD. Dr. Mohamed M. Bumadian Microbiology Department, Benghazi University  We will now look at mechanisms to prevent synthesis of enzymes that are not needed. We have seen that genes, through transcription and translation, direct the synthesis of proteins, many of which serve as enzymes the very enzymes used for cellular metabolism. Because protein synthesis requires a huge amount of energy, regulation of protein synthesis is important to the cell's energy economy. Cells save energy by making only those proteins needed at a particular time. Next we look at how chemical reactions are regulated by controlling the synthesis of the enzymes. Many genes, perhaps 60-80%, are not regulated but are instead constitutive, meaning that their products are constantly produced at a fixed rate.  Repression and Induction We will now look at mechanisms to prevent synthesis of enzymes that are not needed. We have seen that genes, through transcription and translation, direct the synthesis of proteins, many of which serve as enzymes the very enzymes used for cellular metabolism. Because protein synthesis requires a huge amount of energy, regulation of protein synthesis is important to the cell's energy economy. Cells save energy by making only those proteins needed at a particular time. ‫في وقت معين‬  Usually these genes, which are effectively turned on all the time, code for enzymes that the cell needs in fairly large amounts for its major life processes; the enzymes of glycolysis are examples. On the other hand, the production of other enzymes is regulated so that they are present only when needed. ‫التي يتم تشغيلها بشكل فعال طوال الوقت‬ ‫التي تحتاجها الخلية بكميات كبيرة إلى حد ما‬  Trypanosoma, the protozoan parasite that causes African sleeping sickness (disease), has hundreds of genes coding for surface glycoproteins. Each protozoan cell turns on only one glycoprotein gene at a time. As the host's immune system kills parasites with one type of surface molecule, parasites expressing a different surface glycoprotein can continue to grow..‫تقوم كل خلية من الخاليا األولية بتشغيل جين واحد فقط من البروتين السكري في كل مرة‬ ، ‫نظر ألن الجهاز المناعي للمضيف يقتل الطفيليات بنوع واحد من الجزيئات السطحية‬ ‫ًا‬.‫يمكن أن تستمر الطفيليات التي تعبر عن بروتين سكري سطحي مختلف في النمو‬  Repression and Induction Two genetic control mechanisms known as repression and induction regulate the transcription of mRNA and consequently the synthesis of enzymes from them. These mechanisms control the formation and amounts of enzymes in the cell, not the activities of the enzymes. ‫التشكيل‬  Repression o The regulatory mechanism that inhibits gene expression and decreases the synthesis of enzymes is called repression. o Repression is usually a response to the overabundance of an end-product of a metabolic pathway; it causes a decrease in the rate of synthesis of the enzymes leading to the formation of that product. o Repression is mediated by regulatory proteins called repressors, which block the ability of RNA polymerase to initiate transcription from the repressed genes. o The default position of a repressible gene is on. ‫يتم بواسطة‬  Induction o The process that turns on the transcription of a gene or genes is induction. A substance that acts to induce transcription of a gene is called an inducer, and enzymes that are synthesized in the presence of inducers are inducible enzymes. o The genes required for lactose metabolism in E. coli are a well-Known example of an inducible system. o One of these genes codes for the enzyme β-galactosidase, which splits the substrate lactose into two simple sugars, glucose and galactose.  If E. coli is placed into a medium in which no lactose is present, the organisms contain almost no ß-galactosidase; however, when lactose is added to the medium, the bacterial cells produce a large quantity of the enzyme. Lactose is converted in the cell to the related compound allolactose, which is the inducer for these genes; the presence of lactose thus indirectly induces the cells to synthesize more enzyme. The default position of an inducible gene is off.  The Operon Model of Gene Expression Operon is a unit made up of linked genes that is thought to regulate other genes responsible for protein synthesis. Details of the control of gene expression by induction and repression are described by the operon model. Jacob and Jacques Monod formulated this general model in 1961 to account for the regulation of protein synthesis. Structure of the operon. The operon consists of the promoter (P) and operator (O) sites and structural genes that code for the protein. The operon is regulated by the product of the regulatory gene (I).  (A) Lactose Catabolism They based their model on studies of the induction of the enzymes of lactose catabolism in E. coli. In addition to ß-galactosidase, these enzymes include lac permease, which is involved in the transport of lactose into the cell, and transacetylase, which metabolizes certain disaccharides other than lactose.  The genes for the three enzymes involved in lactose uptake and utilization are next to each other on the bacterial chromosome and are regulated together (Figure 8.12). These genes, which determine the structures of proteins, are called structural genes to distinguish them from an adjoining control region on the DNA. When lactose is introduced into the culture medium, the lac structural genes are all transcribed and translated rapidly and simultaneously. We will now see how this regulation occurs. ‫في نفس الوقت‬  Figure 8.12. Lactose-digesting enzymes are produced in the presence of lactose. In E. coli, the genes for the three enzymes are in the lac operon. ß-galactosidase is encoded by lacZ. The lacY gene encodes the lac permease. and lacA encodes transacetylase, whose function in lactose metabolism is still unclear. 1- Repressor active, operon off. The repressor protein (substance) binds with the operator, preventing transcription from the operon (inhibit messenger RNA synthesis). operon Q What causes (reasons) tmnscription of an inducible enzyme? 2- Repressor inactive, operon on. When the inducer allolactose binds to the repressor protein, the inactivated repressor can no longer block transcription. The structural genes are transcribed, ultimately resulting in the production of the enzymes needed for lactose catabolism. transport of lactose Metabolizes into the cell certain disaccharides other than lactose.  In the control region of the lac operon are two relatively short segments of DNA. One, the promoter, is the region of DNA where RNA polymerase initiates transcription. The other is the operator, which is like a traffic light that acts as a go or stop signal for transcription of the structural genes. A set of promoter and operator sites and the structural genes they control define an operon; thus, the combination of the three lac structural genes and the adjoining control regions is called the lac operon. ‫تحديد السيطرة‬  A regulatory gene called the I gene encodes a repressor protein that switches inducible and repressible operons on or off. The lac operon is an inducible operon (see Figure 8.12). In the absence of lactose, the repressor binds to the operator site, thus preventing transcription. If lactose is present, the repressor binds to a metabolite of lactose instead of to the operator, and lactose digesting enzymes are transcribed. In repressible operons, the structural genes are transcribed until they are turned off, or repressed (Figure 8.13).  (B) Synthesis of Tryptophan The genes for the enzymes involved in the synthesis of tryptophan are regulated in this manner (way). The structural genes are transcribed and translated, leading to tryptophan synthesis. When excess tryptophan is present, the tryptophan acts as a corepressor binding to the repressor protein. The repressor protein can now bind to the operator, stopping further tryptophan synthesis.  Figure 8.13 A repressible operon. Tryptophan, an amino acid is produced by anabolic enzymes encoded by five structural genes. Accumulation of tryptophan represses transcription of these genes. preventing further synthesis of tryptophan. The E coli trp operon is shown here. 1- Repressor Inactive, operon on. The repressor is inactive, and transcription and translation proceed, leading to the synthesis of tryptophan. Figure 8.13 2- Repressor active, operon off. When the corepressor tryptophan binds to the repressor protein, the activated repressor binds with the operator, preventing transcription from the operon. Q What causes transcription of a repressible enzyme?  (C) Positive Regulation Regulation of the lactose operon also depends on the level of glucose in the medium, which in turn controls the intracellular level of the small molecule cyclic (cyclic adenosine monophosphate (cAMP)), a substance derived from ATP that serves as a cellular alarm signal. ‫بمثابة إشارة إنذار خلوية‬  Enzymes that metabolize glucose are constitutive, and cells grow at their maximal rate with glucose as their carbon source because they can use it most efficiently (Figure 8.14). When glucose is no longer available, cAMP accumulates in the cell. The cAMP binds to the allosteric site of catabolic activator protein (CAP). o cAMP (Cyclic adenosine monophosphate) is a second messenger important in many biological processes. derivative from ATP and used for intracellular signal transduction in many different organisms. (a) Bacteria growing on glucose as the sole carbon source grow faster than on lactose. Figure 8.14 The growth rate of E. coli on glucose and lactose. Q When both glucose and lactose are present, why will cells use glucose first? (b) Bacteria growing in a medium containing glucose and lactose first consume the glucose and then after a short lag time, the lactose. During the lag time, intracellular cAMP increases, the lac operon is transcribed, more lactose is transported into the cell, and β. galactosidase is synthesized to break down lactose.  Catabolic activator protein (CAP) then binds to the lac promoter, which initiates transcription by making it easier for RNA polymerase to bind to the promoter. Thus transcription of the lac operon requires both the presence of lactose and the absence of glucose (Figure 8.15).  Cyclic AMP is an example of an alarm one, a chemical alarm signal that promotes a cell's response to environmental or nutritional stress. (In this case, the stress is the lack of glucose.). The same mechanism involving cAMP allows the cell to grow on other sugars. Inhibition of the metabolism of alternative carbon sources by glucose is termed catabolite repression (or the glucose effect). When glucose is available, the level of cAMP in the cell is low, and consequently CAP is not bound. 1- Lactose present, glucose scarce (cAMP level high). If glucose is scarce, the high level of cAMP activates CAP, and the lac operon produces large amounts of mRNA for lactose digestion. cyclic adenosine onophosphate catabolic activator protein (CAP). Figure 8.15 Positive regulation of the lac operon. 2- Lactose present, glucose present (cAMP level low). When glucose is present, cAMP is scarce, and CAP is unable to stimulate transcription. Figure 8.15 Positive regulation of the lac operon  Q Will transcription of the lac operon occur in the presence of lactose and glucose? In the presence of lactose and the absence of glucose? In the presence of glucose and the absence of lactose?