5th Course GENE REGULATION 1 Alami UM6SS November 2024 PDF

Gene regulation Pr. Raouf Alami CM1 Module : Cellular and molecular biology Course: Molecular biology Année Universitaire: 2024 - 2025 w...

Gene regulation Pr. Raouf Alami CM1 Module : Cellular and molecular biology Course: Molecular biology Année Universitaire: 2024 - 2025 www.um6ss.ma Learning Objectives By the end of this course, you will be able to : Describe the steps involved in prokaryotic gene regulation Understand the basic steps in operon lactose regulation Explain the roles of activators, inducers, and repressors in gene regulation in prokaryotes Understand some of the mechanisms of eukaryotes gene regulation 2 1 Introduction Gene regulation Gene regulation refers to the mechanisms that act to induce or repress the expression of a gene. Various intrinsic and extrinsic mechanisms regulate gene expression before, during and after transcription. These could include : 1. Structural and chemical changes to the genetic material, Or 2. Binding of proteins to specific DNA elements to regulate transcription, Or 3. Mechanisms that modulate translation of mRNA. 3 Preamble Gene regulation is the process of controlling which genes in a cell's DNA are expressed Different cells in a multicellular organism may express very different sets of genes, even though they contain the same DNA. The set of genes expressed in a cell determines the set of proteins and functional RNAs it contains, In eukaryotes like humans, gene expression involves many steps, and gene regulation can occur at any of these steps. However, many genes are regulated primarily at the level of transcription. 4  Prokaryotes gene expression The DNA of prokaryotes is organized into a circular chromosome, Proteins that are needed for a specific function, or that are involved in the same biochemical pathway, are encoded together in blocks called operons. For example, all of the genes needed to use lactose as an energy source are coded next to each other in the lactose (or lac) operon, and transcribed into a single mRNA. In prokaryotic cells, there are three types of regulatory molecules that can affect the expression of operons: repressors, activators, and inducers. 5  Prokaryotes gene expression Repressors and activators are proteins produced in the cell. Both repressors and activators regulate gene expression by binding to specific DNA sites adjacent to the genes they control. Activators bind to the Repressors bind to promoter site, operator regions Activators increase the transcription of a Repressors prevent transcription of a gene in response to an external stimulus. gene in response to an external stimulus Inducers are small molecules that may be produced by the cell or that are in the cell’s environment. Inducers either activate or repress transcription depending on the needs of the cell and the availability of substrate. 6 Prokaryotes versus Eukaryotes DNA Eukaryotic DNA Prokaryotic DNA Eukaryotic DNA is linear, Bacterial cells have a has telomeres at each double starnd circular end to protect from DNA deterioration 7  Gene Expression in prokaryotes and Eukaryotes Prokaryotic Eukaryotic organisms organisms Lack nucleus Have nucleus DNA is found in the DNA is confined to the nuclear cytoplasm compartment RNA transcription occurs prior to RNA transcription and protein formation, and it takes place protein formation occur in the nucleus. Translation of RNA to almost simultaneously protein occurs in the cytoplasm. Gene expression is regulated at many levels : 1. Chromatin accessibility. Gene expression is 2. Transcription. regulated primarily at the 3. RNA processing. transcriptional level 4. RNA stability. 5. Translation. 6. Protein activity 8 Prokaryotic gene regulation DNA floats freely in the cell cytoplasm. When the resulting protein is no longer needed, transcription stops. The primary method to control what type of protein and how much of each protein is expressed is the regulation of DNA transcription. All of the There subsequent steps occur automatically. are post-translational modifications in prokaryotes, but they are less common Translation in prokaryotes is usually regulated by blocking access to the initiation site 9 Prokaryotes Gene Regulation The amount of product depends on:  rate of mRNA synthesis (transcription),  mRNA degradation,  protein synthesis (translation) Prokaryotes commonly control transcription: 1. Constitutive genes are always expressed  Tend to be vital for basic cell functions (often called housekeeping genes) 2. Regulated genes can be inducible or repressible  Inducible genes are normally off, but can be turned on when substrate is present Example: The lac operon is inducible.  Repressible genes are normally on, but can be turned off when the end product is abundant Example : Tryptophane 10 Key elements in the prokaryotic gene regulation 11 elements in the prokaryotic gene regulation Polycistronic mRNA A cistron is a DNA segment equivalent to a gene Starts at AUG and stops at a codon STOP Bacteria produce a single polycistronic mRNA = single mRNA that contains information to make multiple proteins). Cistron Cistron Cistron 3 1 2 12 elements in the prokaryotic gene regulation Operons What is an operon ? Bacteria encode operons = set of genes under transcriptional control of a single control region (promoter and operator) and a single terminator Operon Control region Structural genes Terminator PromoterOperator An operon is composed of The Operon is controlled by repressors 1. promoter, and activators. 2. an operator, and Repressors and activators are proteins 3. the structural expressed by a specific regulating gene genes. 13 elements in the prokaryotic gene regulation Who discovered the Operon and when ? The regulatory mechanism of the lac operon of Escherichia coli, mRNA was first described by these scientists The Nobel Prize in Physiology or Medicine 1965 was awarded jointly to François Jacob, André Lwoff and Jacques Monod 14 elements in the prokaryotic gene regulation Allosteric protein effector An allosteric protein is one whose physical shape and chemical binding properties change when it is bound by a particular effector molecule. Allosteric proteins: proteins which can have 2 different forms Allosteric inhibitors and activators: Allosteric inhibitors modify the active site of the enzyme so that substrate binding is reduced or prevented. Allosteric activators modify the active site of the enzyme so that the affinity for the substrate increases. 15 elements in the prokaryotic gene regulation There are three ways to control the transcription of an operon: 1.Repressive control, 2.Activator control, 3.Inducible control. (= the effector) 16 elements in the prokaryotic gene regulation Repressor and activator DNA binding capacity can be modulated by effectors. For example In the lac operon, allolactose binds the activator to induce transcription when the operon is under positive control. 17 elements in the prokaryotic gene regulation What is cis and trans variants? Cis-regulatory elements, such as promoters, enhancers, and silencers, are regions of non-coding DNA, which regulate the transcription of nearby genes. Trans-regulatory factors regulate (or modify) the expression of distant genes by combining with their target sequences 18  elements in the prokaryotic gene regulation nducible versus a. Inducible repressible operons operons are turned on in reponse to a metabolite (a small molecule undergoing metabolism) that regulates the operon. E.g. the lac operon is induced in the presence of lactose (through the action of a metabolic by-product allolactose). b. Repressible operons are switched off in reponse to a small regulatory molecule. E.g., the trp operon is repressed in the presence of tryptophan. Lactose Triptophane Lactose shows up ---- the genes express Tryptophan is an amino acid needed for normal growth and for the production and Inducible operator maintenance of the body's proteins, 19  Structure of lac operon The DNA of the lac operon contains (in order from left to right): , 1. Lac I -the lacI gene (regulatory gene) produces a protein that from binding to the operator of the operon. This protein can only be removed when allolactose binds to it. 2. CAP binding site -Catabolite activator protein- 3. Promoter (RNA polymerase binding site), 4. Operator (which overlaps with promoter), 5. LacZ gene, 6. LacY gene, 7. Lac A gene. 20  Structure of lac operon Lac I gene b The LacI gene is a regulatory 1 gene that codes for repressor. In other words, it codes 2 for the respressor of the Lac-operon. LacI is always transcribed. 3 When the repressor binds to the operator, the Lac genes can't be 21 transcribed.  CAP site Catabolite activator protein site CAP site mRNA Lac Repressor b Galactosidase Protein (R) Permease Transacetylase Catabolite activator protein (CAP) must bind to cAMP to activate transcription of the lac operon by RNA polymerase. CAP is a transcriptional activator 22 Structure of lac operon Promoter Promoter – a nucleotide sequence that enables a gene to be transcribed. The promoter is where the RNA polymerase attaches CAP site mRNA Lac Repressor b Galactosidase Protein (R) Permease Transacetylase 23 Structure of lac operon Operator Operator – a segment of DNA to which a repressor binds. It is a segment between the promoter and the genes of the operon. CAP site mRNA Lac Repressor b Galactosidase Protein (R) Permease Transacetylase 24 Structure of lac operon Structural genes lacZ, lacY, and lacA CAP site mRNA Lac Repressor b Galactosidase Protein (R) Permease Transacetylase The lac operon contains three genes: lacZ, lacY, and lacA. These genes are transcribed as a single mRNA, under control of one promoter. 25  Structure of the lac operon Splits lactose into Regulatory DNA sequences monosaccharides a positive Transport Negative regulatory lactose into the regulatory site cell RNA pol sitesite CAP = catabolite activator protein Role of these gene : import lactose into cells and break it down for use as a food source. https://opentextbc.ca/biology2eopen stax/chapter/prokaryotic-gene-regula 26 tion/  Structure of lac operon The lac operon encodes the genes for the transport of external lactose into the cell and its conversion to glucose and galactose. There is a regulatory gene lacI preceding the lac operon. lacI is responsible for producing a repressor (R) protein. Cytoplasm Repres Beta Perme sor Gal ase Transport inside the cell Gluco b Gal Allola Lacto se c se Lacto se 27 Prokaryotes gene regulation Lac Operon Adapting to the environment monosaccharid disaccharide E. coli can use either : glucose, which is a monosaccharide, or lactose, which is a disaccharide Lactose can be an excellent meal for E. Coli bacteria. However, they'll only up-take lactose when other, better sugars – like glucose – are unavailable. The genes in the lac operon encode proteins that allow the bacteria to use lactose as an energy source. 28  Adapting to the environment Lac Operon Four environmental situations are possible Adapting to the environment 1 When glucose is present 2 When glucose is present and lactose is absent and lactose is present When glucose is absent 3 When glucose is absent and lactose is absent and lactose is present 4 29  When the genes are transcribed? When glucose is present When glucose is present and lactose is present the and lactose is absent the E. coli does not produce β- E. coli does not produce galactosidase. β-galactosidase. Expression not needed Expression not needed When glucose is absent When glucose is absent and lactose is absent the E. and lactose is present coli does not produce β- the E. coli does produce galactosidase. Expression is needed β-galactosidase. Expression not needed 30  Adapting to the environment This positive control system works as follows: When there is no glucose available for cellular metabolism but if lactose is available in a media, the lactose is transported into the cell by the permease. Once inside the cell, lactose is then broken down (by β-galactosidase) into : 1. glucose, 2. galactose, and 3. Allolactose The allolactose feeds back to bind with the lactose repressor and enable the transcription process which completes the positive feedback loop. 31  Adapting to the environment Inducer = allolactose The inducer in the lac operon is allolactose. If lactose is present in the medium, then a small amount of it will be converted to allolactose by a few molecules of β-galactosidase that are present in the cell. Allolactose binds to the repressor and decreases the repressor's affinity for the operator site. However, when lactose and glucose are both available in the system, the lac operon is repressed. This is because glucose actively prevents the induction of lacZYA 32 https://youtu.be/iPQZXMKZEfw 33 When lactose is absent, the lac repressor binds to the oper This prevent RNApol moving forward. No Transcription No mRNA The bacteria will stop making the enzymes that degrade the lactose (Saving energy) https://opentextbc.ca/biology2eopenstax/chapter/prokaryotic-ge e-regulation/  Inducer = allolactose When Lacrose is available Allolactose a form of lactose binds to the repressor and makes if leave the oper This will allow RNA Pol to move forward and transcribe the 3 genes Lactose Allolactose = isoforme of lactose = similar to lactose 35 When Glucose present and lactose Absent 36 2- When glucose is present and lactose is present 37 3 When glucose is absent and lactose is absent 38  4- When glucose is absent lactose is present E coli produce a hunger signal cAMP = Cyclic adenosine monophosphate called cAMP The cAMP (cyclic AMP) attaches to CAP allowing it to bind to the DNA. CAP helps RNApol binds to promoter , resulting in high levels of transcription 39 Two regulatory proteins are involved in the regulation of the operon: 1. the Lac repressor and 2. the CAP activator, These two proteins bind to DNA. 3 situations are there: 1. In the presence of lactose, the repressor is inhibited by lactose and cannot bind to the operator. Lactose is therefore an inducer and induces the translation of proteins involved in its metabolism. 2. In the absence of glucose, the CAP protein binds to DNA and activates the genes of the operon. 3. In the presence of glucose (better source of energy than lactose), gene expression is inhibited even in the presence of lactose because CAP cannot bind to DNA. The lactose operon is therefore an inducible operon. 41  trp operon Tryptophan is an amino acid that E coli can manufacture The trp operon is : 1.expressed (turned "on") when tryptophan levels are low 2.repressed (turned "off") when they tryptophan levels are If tryptophan high. in the is present trp operon is switched "off." environment, then E. coli bacteria don't need to synthesize it, When tryptophan availability is low, the operon is switched "on," tryptophan is produced. 42  Summary An inducer Is a molecule that must be present to interact wih the repressor or the activator to either stop transcription or increase it 1- Lac operon The inducer in the lac operon is allolactose. If lactose is present in the medium, then a small amount of it will be converted to allolactose. Allolactose binds to the repressor and decreases the repressor's affinity for the operator site. expression Genes 2- trp operon tryptophan is an inducer The trp operon is regulated by the trp repressor. When the trp repressor is bound to tryptophan (inducer), the trp repressor blocks expression of the operon. Genes silenced 43  Gene regulation in eukaryotes Gene regulation makes cells different Gene regulation is how a cell controls which genes, out of the many genes in its genome, are "turned on" (expressed). Or turned off (repressed) These different patterns of gene expression cause that all the various cell types to make each cell type uniquely specialized to do its job. Cell specialization 44 Cell types in the human body There are over 200 different cell types in the human body. Each type of cells is specialized to carry out a particular function, either solely, Fewbut usually by examples formingcells of human a particular tissue. Red blood cells Erythrocytes Granulocytes (neutrophils, eosinophils, basophils) White blood cells Agranulocytes (monocytes, lymphocytes) Neurons Nerve cells Neuroglial cells Muscle cells Skeletal, Cardiac, Smooth Osteoblasts, Osteoclasts, Bone cells Osteocytes , Lining cells Keratinocytes, Melanocytes , Skin cells Merkel cells , Langerhans cells White adipocytes, Brown Fat cells adipocytes 45 Sex cells Spermatozoa, Ova Cell types in the human body Gene regulation is the origin of cell specialization Hepatocytes Nerve cell Red blood cells 46  How cells get specialized? Eukaryotic gene expression can be regulated at many stages Expression of genes in eukaryotes can be controlled at various stages, from the availability of DNA to the production of mRNAs to the translation and processing of proteins. Eukaryotic gene expression involves many steps, and almost all of them can be regulated. We can list at least 6 levels of gene regulation: Reference: Reece, J. B., Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., and Jackson, R. B. (2011). Figure 18.6. Stages in gene expression that can be regulated in eukaryotic cells. In Campbell Biology (10th ed., p. 365). San Francisco, CA: Pearson. 47 Eukaryotic gene expression can be regulated at many stages Gene Expression Can Be Regulated at Any Stage 1. Chromatin accessibility. More open or “relaxed” chromatin makes a gene more available for transcription. 2. Transcription. Transcription is a key regulatory point for many genes. Sets of transcription factor proteins bind to specific DNA sequences in or near a gene and promote or repress its transcription into an RNA. 3. RNA processing. All the mRNA maturation can be regulated (Splicing, capping, and addition of a poly-A tail). in addition, many mRNAs may be made from the same gene by alternative splicing. 4. RNA stability. The lifetime of an mRNA molecule in the cytosol affects how many proteins can be made from it. 5. Translation. Translation of an mRNA may be increased or inhibited by regulators. 48 6. Protein activity. Proteins can undergo a variety of modifications,  Eukaryotic Gene Regulation gene expression in Eukaryotic is more complex than in prokaryotic because the processes of transcription and translation are physically separated. Eukaryotic cells can regulate gene expression at many different levels. Eukaryotic gene expression begins with control of access to the DNA. Transcriptional access to the DNA can be controlled in two general ways: 1. chromatin remodeling (associated with chromosomal histones) https://www.nature.com/scitable/topicpage/gene-expressio 2. DNA methylation (genen-14121669/ 2010 Nature Education All rights reserved silencing)  Eukaryotic Gene Regulation Why gene regulation involves more steps in eukaryotes? 1. Eukaryotes cells contains much more amount of DNA 2. The mRNA of most eukaryotes must be spliced 3. Genetic information in eukaryotes is carried on many chromosomes and these chromosomes are enclosed within a double-membrane-bound nucleus 4. Eukaryotes mRNAs can have a wide range of half-lives. Rapid decay of prokaryotes mRNAs allow them to adapt quickly to the environement. 5. In eukaryotes, translation rates can be modulated, as well as the proteins are processed and modified 50  Eukaryotic Gene Regulation ting Regulatory Sequences: Promoters and Enhancer Enhancers / silencer SilencerEnhancers v Enhancers 1. Are short DNA sequence (50–1500 bp) 2. Can be bound by proteins 3. Increase the transcription of a Gene particular gene Intron Exon Silencer 1. are short DNA sequence 2. capable of binding transcription regulation factors, (called repressors) 3. Decrease or inhibit  Eukaryotic Gene Regulation cting Regulatory Sequences and transcription factors For gene transcription to occur, a number of transcription factors must bind to DNA regulatory sequences. This collection of transcription factors, in turn, recruit intermediary proteins such as cofactors that allow efficient recruitment of RNA polymerase. The loop will allow the proteins that bind to the enhancer to interact with protein attached to TATA box DNA 52  Eukaryotic Gene Regulation Transcription factor The most common General TFs are : TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and domain, TFs contain at least one DNA-binding TFIIH. which attaches to a specific sequence of DNA adjacent to the genes that they regulate. TFs are grouped into classes based on their DBDs There are approximately 2800 proteins in the human genome that contain DNA-binding domains amino carboxylic terminus acid terminus DNA-binding domain signal- Activation sensing domain domain Schematic diagram of the amino acid sequence of a TF Note: The order of placement and the number of domains may differ in various types of TFs. 53 https://en.wikipedia.org/wiki/Transcription_factor#DNA-  Transcription factors Transcription factors are often classified based on the tertiary structure of their DNA-binding domains (were initially described in the 1980s) The major TF families in eukaryotes are: basic helix-loop-helix basic-leucine zipper (bZIP) C-terminal effector domain of the bipartite response regulators Helix-loop-helix C2H2 zinc finger proteins Basic leucine zipper 54 What are the major transcription factor families? DNA-binding domain 55  Eukaryotic Gene Regulation Post transcription regulation Even after a gene has been transcribed, gene expression can still be regulated at various stages.  Some transcripts can undergo alternative splicing, making different mRNAs and proteins from the same RNA transcript.  Some mRNAs are targeted by microRNAs, small regulator RNAs that can cause an mRNA to be chopped up or block translation.  A protein's activity may be regulated after translation, for example, through removal of amino acids or addition of chemical groups. RNA processing, such as splicing, capping, and poly-A tail addition Messenger RNA (mRNA) translation and lifetime in the cytosol Protein modifications, such as addition of chemical groups 56  Eukaryotic Gene Regulation Alternative splicing Different cell types may express different regulatory proteins, so different exon combinations can be used in each cell type, leading to the production of different proteins. Alternative splicing is not a random process. Instead, it's typically controlled by regulatory proteins. The proteins bind to specific sites on the pre-mRNA and "tell" the splicing factors which exons should be used. 57  Eukaryotic Gene Regulation Small regulatory RNAs A recently discovered class of regulators, Two key determinants of how much protein is made from an mRNA are : 1. its "lifespan" (how long it floats around in the cytosol) 2. how readily the translation machinery, such as the ribosome, can attach to it. microRNAs microRNAs (miRNAs) were among the first small regulatory RNAs to be discovered. 58  Eukaryotic Gene Regulation mRNA Editing In some instances, an mRNA will be edited, changing the nucleotide composition of that mRNA. What is the main advantage of RNA editing? RNA editing may be advantageous for adaptation because it contributes to transcriptome diversity, and can be used to fine-tune protein function in response to the environment. An example in humans is the apolipoprotein B mRNA, which is edited in some tissues, but not others. The editing creates an early stop codon, which, upon translation, 59 produces a shorter protein.  Eukaryotic Gene Regulation Post translation regulation After RNA translation into protein, regulation of pre-made proteins can help cells respond to stimuli or change their behaviors in a quick, sharp way. It’s a simple chemical modification, without the need for time- consuming transcription and translation. This regulatory mechanisms act as an "editing process" of the newly produced protein, such as : 1. removal of amino acids, or 2. addition of a chemical modification – can lead to a change in its activity or behavior. Why post translation steps are sometimes needed for the cell? -Save time and energy- 60  Eukaryotic Gene Regulation Post translation regulation Addition or removal of chemical groups may regulate protein activity or the length of time a protein remains in the cell before it undergoes "recycling." Chemical modifications can also determine where a protein is found in the cell  in the nucleus  in cytoplasm,  attached to the plasma membrane. 61  Eukaryotic Gene Regulation Post translation regulation Phosphorylation One of the most common post-translational modifications is phosphorylation, in which a phosphate group is attached to a protein. The effect of phosphorylation varies from protein to protein: some are activated by phosphorylation, others are deactivated, others yet simply change their behavior (interacting with a different partner, or going to a different part of the cell). 62  Eukaryotic Gene Regulation Post translation regulation Ubiquitination Proteins can be tagged for degradation by the addition of a chemical marker called ubiquitin. Ubiquitination is an important way of controlling the persistence of a protein in the cell. Ubiquitin is a small, 76-amino acid, regulatory protein that was discovered in 1975. It's present in all eukaryotic cells, Ubiquitin regulates the stability and functional activity of proteins. participating in both the synthesis of new proteins and the destruction of defective proteins. 63  Gene regulation, continued on the next course,……. 64