Regulation of Gene Expression in Prokaryotes PDF

Course: General Genetics AGN 101 Regulation of Gene Expression in living organisms BY PROF. DR. MOHAMED EL-AWADY Learning Objectives By the end of this section, you will be able to:  Discuss why every cell does not express all of its genes.  Describe some ma...

Course: General Genetics AGN 101 Regulation of Gene Expression in living organisms BY PROF. DR. MOHAMED EL-AWADY Learning Objectives By the end of this section, you will be able to:  Discuss why every cell does not express all of its genes.  Describe some major differences between prokaryotic and eukaryotic gene regulation.  Describe the steps involved in prokaryotic gene regulation.  Explain the roles of activators, inducers, and repressors in gene regulation  Overview of Regulation of Gene Expression - For a cell to function properly, necessary proteins must be synthesized at the proper time. All cells control or regulate the synthesis of proteins from information encoded in their DNA. - The process of “turning on” a gene to produce mRNA and protein is called gene expression. - Whether in a simple unicellular organism or a complex multi- cellular organism, each cell controls when its genes are expressed, how much of the protein is made, and when it is time to stop making that protein because it is no longer needed. - It is more energy efficient to turn on the genes only when they are required. - In addition, only expressing a subset of genes in each cell saves space because DNA must be unwound from its tightly coiled structure to transcribe and translate the DNA. - Cells would have to be enormous if every protein were expressed in every cell all the time. - The regulation of gene expression conserves energy and space. - The control of gene expression is extremely complex. - Malfunctions in this process are detrimental to the cell and can lead to the development of many diseases, including cancer.  Prokaryotic versus Eukaryotic Gene Expression Since prokaryotic organisms are single-celled organisms that lack a cell nucleus, their DNA floats freely in the cell’s cytoplasm. - When a particular protein is needed, the gene that codes for it is transcribed in mRNA, which is simultaneously translated into protein. When the protein is no longer needed, transcription stops. - As a result, the primary method to control how much of each protein is expressed in a prokaryotic cell is the regulation of transcription. Eukaryotic cells, in contrast, have intracellular organelles that add to their complexity. In eukaryotic cells, the DNA is contained inside the cell’s nucleus, where it is transcribed into mRNA. - The newly synthesized mRNA is then modified and transported out of the nucleus into the cytoplasm, where ribosomes translate the mRNA into protein. - The processes of transcription and translation are physically separated by the nuclear membrane; transcription occurs only within the nucleus, and translation occurs only in the cytoplasm. - The regulation of gene expression in eukaryotes can occur at all stages of the process. Prokaryotic transcription and translation occur simultaneously in the cytoplasm, and regulation occurs at the level of transcription. In eukaryotes, transcription and translation are physically separated, and gene expression is regulated at many different levels.  Prokaryotic organisms Eukaryotic organisms Lack nucleus Contain nucleus DNA is found in the cytoplasm DNA is in the nucleus Transcription occurs in the nucleus Transcription and translation occur prior to translation, which occurs in almost simultaneously the cytoplasm. Gene expression is regulated at many Gene expression is regulated primarily levels: epigenetic, transcriptional, at the transcriptional level nuclear shuttling, post- transcriptional, translational, and post-translational Some of the differences in the regulation of gene expression between prokaryotes and eukaryotes.  Prokaryotic Gene Regulation 1- Housekeeping (constitutive) and inducible genes The DNA of prokaryotes is organized into a circular chromosome supercoiled in the nucleoid region of the cell cytoplasm. Bacterial genomes usually contain several thousand different genes. - Some of the gene products are required by the cell under all growth conditions and are called housekeeping genes. These include the genes that encode such proteins as DNA polymerase, RNA polymerase, and DNA gyrase. Housekeeping genes must be expressed at some level all of the time. Frequently, as the cell grows faster, more of the housekeeping gene products are needed. Even under very slow growth, some of each housekeeping gene product is made. Inducible genes Many other gene products are not needed all of the time but only required under specific growth conditions. These include enzymes that synthesize amino acids, break down specific sugars, or respond to a specific environmental condition such as DNA damage. - These genes are frequently expressed at extremely low levels, or not expressed at all when they are not needed and yet made when they are needed. - The question is …………………How bacteria regulate the expression of their genes so that the genes that are being expressed meet the needs of the cell for a specific growth condition?  2- Levels of gene expression regulation in prokaryotes Gene regulation can occur at three possible levels in the production of an active gene product. First, transcriptional regulation. The transcription of the gene can be regulated. as When the gene is transcribed and how much it is transcribed influences the amount of gene product that is made. Second, translational regulation If the gene encodes a protein, it can be regulated at the translational level. This is how often the mRNA is translated influences the amount of gene product that is made. Third, gene products can be regulated after they are completely synthesized by either post-transcriptional or post- translational regulation mechanisms. - Both RNA and protein can be regulated by degradation to control how much active gene product is present. 3- Operon concept The DNA of prokaryotes (bacteria and archaea) is organized into a circular chromosome that resides in the cell’s cytoplasm. To save energy, proteins that are needed for a specific function, or that are involved in the same biochemical pathway (enzymes that catalyze the many steps in a single biochemical pathway), are often encoded together within the genome in blocks called operons. The genes in an operon are transcribed together under the control of a single promoter into a single mRNA molecule (a polycistronic transcript). This allows the genes to be controlled as a unit, either all are expressed (all is needed at the same time), or none is expressed (none is needed). - Each operon needs only one regulatory region, including a promoter, where RNA polymerase binds for initiating mRNA synthesis, and an operator, where other regulatory proteins bind and a terminator indicating end of mRNA synthesis. Operon is a unit of expression and regulation. In bacteria, cistrons or structural genes, producing enzymes of a metabolic pathway are organized in a cluster whose functions are related. Polycistronic genes of prokaryotes along with their regulatory genes constitute a system called operon.  4- The presence of Regulatory Proteins In bacteria the expression of genes is controlled by extracellular signals often present in the medium in which bacteria are grown. These signals are carried to the genes by regulatory proteins. 1- Negative Regulators or Repressors: Repressors are proteins that suppress transcription of a gene in response to an external stimulus. The repressor or inhibitor protein binds to the target site (operator) on DNA. These block the RNA polymerase enzyme from binding to the promoter, thus preventing the transcription. The repressor binds to the site where it overlaps the polymerase enzyme. Thus, activity of the genes is turned off. It is called negative control mechanism. Co-Repressors: are small molecules bind to the repressor and enables it to bind to DNA causing repression of gene expression 2- Positive Regulators or activators: Activators are proteins that increase the transcription of a gene in response to an external stimulus. It activate the transcription by the promoter, the activator helps polymerase enzyme to bind to the promoter Inducers: are small molecules that act as an anti-repressor or anti- inhibitor and thereby activating the genes. Thus, the genes are switched on.  5- Inducible and Repressible pathways Escherichia coli is an excellent model organism for studying gene regulation. They can switch on and switch off expression of certain genes depending upon environment or phase of life cycle like gene replication, cell division, etc. 1- Inducible pathways (usually catabolism pathways): It was observed that bacteria synthesize lactose-metabolizing enzymes only when lactose was present in the medium. These enzymes were therefore called as adaptive or facultative enzymes. Later, that terminology was replaced with inducible enzyme as the production gets induced only when the inducer-like lactose is present. The pathway is then said to be inducible (The other enzymes which are always present are called constitutive enzymes). 2- Repressible pathways: (usually anabolism pathways): A contrasting system also exists, where the presence of gene product inhibits the gene expression. - Tryptophan is an amino acid which gets synthesized by the cell. If tryptophan is present in sufficient amount, the cell does not need to synthesize it anymore. Therefore, it inhibits the anabolic pathway which leads to tryptophan synthesis. Therefore, the end product of the pathway tryptophan acts as a repressor. Therefore, the pathway is then said to be repressible.  6- Inducible and repressible pathways can be controlled by positive and negative regulation. - In the negative regulation mode: The RNA production continues as default unless it is being shut off by a regulator molecule, that regulator molecule is called as repressor. - Sometimes, the repressor can not bind to DNA by it self, so another small molecule binds to the repressor and enables it to bind to DNA causing repression of gene expression. - This small molecule is called as co-repressor. - Other mechanism for gene repression is an inhibitor binding to an activator so that positive regulation by activator does not occur. - Both corepressor and inhibitor lead to stop the gene repression. In the positive regulation mode: - The gene expression continues when a regulator molecule directly stimulates RNA production. - In this case, the regulator molecule is called as activator. - Both inducible and repressible systems could be controlled by the combination of positive and negative control. Binding sites on a genetic regulatory protein. In these examples, a regulatory protein has two binding sites: one for DNA and the other for a small effector molecule. The conformational changes in the regulatory protein are brought about by the binding of small effector molecule, leading to changes in its DNA-binding site. a) Regulation of operon by repressor in presence and absence of inducer. b) Regulation of operon by activator in presence and absence of inducer. c) Regulation of operon by repressor in presence and absence of co- repressor. d) Regulation of operon by activator in presence and absence of inhibitor 8- Lactose Operon or Lac Operon (An Inducible Operon) -In 1961 Francois Jacob and Jacques Monod proposed operon model for the regulation of gene expression in E. coli. The synthesis of enzyme (3-galactosidase has been studied in detail. This enzyme causes the breakdown of lactose into glucose and galactose. In the absence of lactose, β-galactosidase is present in negligible amounts. As soon as lactose is added from outside, the production of β- galactosidase increases thousand times. As soon as the lactose in consumed, the production of the enzyme again drops. - The enzymes whose production can be increased by the presence of the substrate on which it acts are called inducible enzyms. - Addition of lactose to the culture medium of E. coli induces the formation of three enzymes (5-galactosidase, permease and transacetylase, which degrade lactose into glucose and galactose. - The genes, which code for these enzymes lie in a cluster and are called cistrons or structural genes. They are transcribed simultaneously into a single mRNA chain (polycistronic), which has codons for all the three enzymes. - The functioning of structural genes to produce mRNA is controlled by regulatory genes. - Regulatory genes consist of Regulator I, Promoter P and a control gene called operator gene O.  Structural organization of lac operon. Regulatory lacI genes has its own lacI promoter. Lac operon has a catabolite activator protein (CAP) site (purple), lac promoter (lacP, light orange), lac operator (lacO, green), structural genes (lacZYA, blue), and lac terminator (gray)  Structural organization of lac operon A- Structural genes B- Regulatory genes A- Structural genes: Three protein-encoding genes lacZ, lacY, and lacA. 1- lacZ encodes for β-galactosidase. This enzyme cleaves lactose into glucose and galactose. the enzyme can catalyze the transgalactosylation of lactose to allolactose, and the allolactose can be cleaved to the monosaccharides. It is allolactose that binds to lacZ repressor and creates the positive feedback loop that regulates the amount of β-galactosidase in the cell. 2- lacY encodes for an integral cytoplasmic membrane protein, lactose permease. This protein helps in active uptake of lactose. 3- lacA encodes galactoside transacetylase which modifies lactose and other lactose analogues by attaching hydrophobic acetyl groups. Attachment of acetyl group helps lactose to diffuse out of the cell, preventing toxicity caused by excessive lactose. B- Regulatory gene: lacI gene has its own promoter which is constitutively expressed at a low level. It encodes for lac repressor which binds to the operator of the lac operon.  How dose lac operon work? 1- When lactose is not present, the proteins to digest lactose are not needed. Therefore, a repressor binds to the operator and prevents RNA polymerase from transcribing the operon. 2- When the inducer (lactose) in supplied from outside (is present), allolactose binds to the repressor and removes it from the operator. RNA polymerase is now free to transcribe the genes necessary to digest lactose. 3- However, the story is more complex than this. Since E. coli prefers to use glucose for food, the lac operon is only expressed at low levels even when the repressor is removed. Transcription of the lac operon only occurs when lactose is present. Lactose binds to the repressor and removes it from the operator.  Positive control of lace operon But what happens when ONLY lactose is present? - Now the bacterium needs to ramp up production of the lactose- digesting proteins. - It does so by using an activator protein called catabolite activator protein (CAP). - When glucose levels drop, cyclic AMP (cAMP) begins to accumulate in the cell. - cAMP binds to (CAP) and the complex binds to the lac operon promoter. - This increases the binding ability of RNA polymerase to the promoter and ramps up transcription of the genes. When there is no glucose, the CAP activator increases transcription of the lac operon. However, if no lactose is present, the operon is not activated. In summary, for the lac operon to be fully activated, two conditions must be met. First, the level of glucose must be very low or non-existent. Second, lactose must be present. Only when glucose is absent and lactose is present will the lac operon be transcribed maximally. This makes sense for the cell, because it would be energetically wasteful to create the proteins to process lactose if glucose was plentiful or lactose was not available (Table). Summary of signals that induce or repress transcription of the lac operon.  Mutations in lac operon genes In the 1950s, Jacob, Monod, and Pardee identified rare mutant strains of bacteria which had abnormal lactose metabolism. - One type of mutant-designated lacI resulted in the constitutive expression of lac operon. Even when the lactose was absent, lac operon in these mutants continued to express. - The exact mode of action of lac repressor was not known at that time. It was thought to produce an activator for the operon which kept it transcriptionally active throughout. 1- Mutations in Regulator Gene: Lac repressor encoded by the lacI gene has two binding sites: one for binding the DNA at the operator site and other for binding allolactose. - There are lacI mutants which either fail to produce lac repressor or the repressor is unable to bind to the operator site. Hence, the lac operon remains constitutively active both in presence and absence of lactose. - There are other regulatory mutations known for the lacI gene. lacIs mutation produces repressor that cannot bind allolactose. Hence, it always remains bound to the operator Even if lactose is present, the operon cannot be induced creating a super-repressed state. Mutation in Structural Genes: Mutant strains which had lost the ability to synthesize β- galactosidase or permease were identified and were mapped to lacZ and lacY structural genes, respectively. These mutations led to changes in the amino acid sequence and structure of these proteins which caused loss of function in most cases.  9- The trp Operon: A Repressible Operon - Like all cells, bacteria need amino acids to survive. Tryptophan is one amino acid that the bacterium E. coli can either ingest from the environment or synthesize. - When E. coli needs to synthesize tryptophan, it must express a set of five proteins that are encoded by five genes. These five genes are located next to each other in the tryptophan (trp) operon. - When tryptophan is present in the environment, E. coli does not need to synthesize it, and the trp operon is switched off. - However, when tryptophan availability is low, the trp operon is turned on so that the genes are transcribed, the proteins are made, and tryptophan can be synthesized. - The genes present in this operon are responsible for encoding enzymes involved in the synthesis of tryptophan. - This operon contains five enzyme-encoding genes called as trpE, trpD, trpC, trpB, and trpA, which are involved in tryptophan biosynthesis. - A part from these, there are two genes, trpR and trpL, that play a role in regulation of trp operon. - TrpL is part of trp operon, and trpR acts as a separate transcriptional unit having its own promoter. trpR encodes for trp repressor protein. The five genes that are needed to synthesize tryptophan in E. coli are located next to each other in the trp operon. Overview of trp operon. In absence of tryptophan, operon transcription occurs. In presence of high tryptophan, operon is repressed  How dose trp Operon work? A DNA sequence called the operator is located between the promoter and the first trp gene. The operator contains the DNA code to which the repressor protein can bind. - The repressor protein is regulated by levels of tryptophan in the cell. - When tryptophan is present in the cell, two tryptophan molecules bind to the trp repressor. This causes the repressor to change shape and bind to the trp operator. Binding of the tryptophan– repressor complex at the operator physically blocks the RNA polymerase from binding, and transcribing the downstream genes. - Thus, when the cell has enough tryptophan, it is preventing from making more. -Binding of corepressor to the repressor brings a conformational change in the repressor, and hence it can bind to the operator. Binding of repressor hinders the binding of RNA polymerase to the promoter, and hence trp operon is shut off. - The anabolic end product of the trp operon, tryptophan, itself acts as the repressor for the operon.  Concept of Attenuation We learned that trp repressor binds to the operator site only in the presence of corepressor tryptophan. Hence, in the presence of tryptophan, the operon for tryptophan synthesis is shut off. - In 1970s, Yanofsky and colleagues observed mutant strains of bacteria which lacked trp repressor but could still inhibit trp operon in the presence of high tryptophan concentration. - They also found mutations where trpL gene which codes for the leader sequence was missing and high expression of trp operon was observed. - Studies on these mutant strains led to the understanding of another mode of trp operon regulation called attenuation. During attenuation, transcription begins but is terminated before the complete mRNA is made. - A short stretch of mRNA is transcribed, and it terminates shortly past the trpL gene. - The transcription is terminated before the transcription of structural genes; hence, attenuation stops production of tryptophan. - The attenuation occurs due to the presence of attenuator sequence. - This sequence is present immediately downstream from the operator site. - The first gene in trp operon is trpL gene. - The mRNA of trpL gene codes for 14 amino acids that form the leader peptide. Sequence of the trpL mRNA produced during attenuation. trpL mRNA has self complementary regions which base-pairs with each other. Regions 1 and 2, 2 and 3, and 3 and 4 have possible base pairing. One region can base-pair with the other only once. So if 2 has hydrogen bonded with 1, it cannot base-pair with 3. Similarly, if region 3 has hydrogen bonded with 4, it cannot base-pair with 2. The last U in the purple attenuator sequence is the last nucleotide that would be transcribed if attenuation is occurring - The leader peptide contains two tryptophan's codons. - These two tryptophan codons act as sensor for tryptophan levels in the cell. - If tryptophan levels are high, then only the tRNA gets charged with tryptophan, and leader peptide is synthesized. - In case tryptophan is less, the tRNA doesn’t get charged with tryptophan, and the leader peptide synthesis slows down. - Another important feature of leader mRNA is that it has self- complementary regions which base-pairs with each other to form stem loops. - Region 2 is complementary to region 1 and also to region 3. Region 3 is complementary to region 2 as well as to region 4. Therefore, three stem loops are possible: 1–2, 2–3, and 3–4. - One region can take part in only one stem loop formation. - So if region 2 pairs with 1, it cannot base-pair with region 3. Alternatively, if region 2 base-pairs with 3, then region 3 cannot base- pair with region 4. - The 3–4 stem loop together with U-rich attenuator forms an intrinsic terminator and leads to ρ-independent termination. The amount of tryptophan regulates the formation of 3–4 stem loop. In bacteria, generally, translation and transcription occur together. 1- At normal level of tryptophan, As the transcription of trpL gene continues, region 1 rapidly hydrogen bond with region 2, and region 3 is not able to base-pair with region 2 and can therefore hydrogen bond with region 4. Therefore, the attenuation occurs just after the transcription of trpL gene. 2- When tryptophan concentration is low, trp-charged tRNA is not formed and is enough amount to support translation. Ribosome halts at Trp codons of leader trpL gene as it waits for charged tRNAtrp. When this occurs, region 1 is covered by ribosome and cannot base- pair with region 2. Therefore, region 2 base-pairs with region 3. As region 3 cannot base-pair with region 4, 3–4 stem loop is not formed and attenuation does not occur. Therefore, the tryptophan operon is successfully transcribed. Mechanism of attenuation of trp operon by stem loop formation of trpL mRNA. Attenuation occurs in part (a) and (c) due to formation of a 3–4 stem loop. (a) No Translation, 1-2 and 3-4 stem loops form. b) Low Tryptophan Levels, 2-3 stem-loop forms c) High Tryptophan levels, 3-4 stemloop form) Attenuation regulates transcription of many other amino acid- synthesizing operon. In all these operons, the leader peptide contains the amino acid which is synthesized by the enzymes coded by the operon. For example, histidine operon has seven histidine codons in the leader sequence.