BIOL 23373 - General Genetics Lecture 16 - Gene Regulation I PDF

BIOL 23373 – General Genetics Fall 2024 Lecture 16 Gene Regulation I Announcements Bonus Quiz 5 was due before class today (Monday, Sept. 30). Exam 2 is in class on Wed., Oct. 9. Exam prep resources will be posted to Bb this week. Those with CEA a...

BIOL 23373 – General Genetics Fall 2024 Lecture 16 Gene Regulation I Announcements Bonus Quiz 5 was due before class today (Monday, Sept. 30). Exam 2 is in class on Wed., Oct. 9. Exam prep resources will be posted to Bb this week. Those with CEA accommodations need to make their appointment to take exam at testing center. Tutoring Tutoring @ the CORD offers 1-on-1 and small group assistance in over 100 U of A courses. Students can meet with tutors in person or online by scheduling an appointment or accessing drop-in services. Learn more about Tutoring services and book an appointment at: https://success.uark.edu/academic-initia tives/tutoring.php Corresponding Readings Chapter sections: 16.1-16.5 Gene Regulation Overview Gene regulation refers to the ability of cells to control their level of gene expression Majority of genes are regulated to ensure that proteins are produced at the correct time and place, and in the correct amount Saves energy by producing as needed Constitutive genes are unregulated and have essentially constant levels of expression Their proteins are involved in routine tasks and basic cell functions (i.e., housekeeping genes) Prokaryotes vs. Eukaryotes 6 Prokaryotic Gene Regulation Allows cells to respond to changes in the environment For example, Escherichia coli and lactose When lactose is available, two proteins are made: lactose permease – transports lactose into the cell β-galactosidase – breaks down lactose When lactose levels drop, the proteins are no longer made Prokaryotic Gene Regulation Gene Bacterial gene regulation Transcription Regulation Most commonly occurs at mRNA the level of transcription Translation Can also control rate of translation Protein Can be regulated at protein Post- translation or post-translation level as well Functional protein (a) Bacterial gene regulation 8 Eukaryotic Gene Regulation Necessary to produce different cell types in an organism – cell differentiation All of the organism’s cells contain the same genome but express different proteomes due to gene regulation Different proteins and different amounts of the same protein 118 μm 179 μm 71 μm (a) Skeletal muscle cell (b) Neuron (c) Skin cell Eukaryotic Gene Regulation Gene Eukaryotic gene regulation Transcription Regulation Transcriptional regulation pre-mRNA common RNA processing RNA processing mRNA Translation Translation Post-translation Protein Post- translation Functional protein (b) Eukaryotic gene regulation Regulation of Gene Expression in Bacteria Genes needed for responding to changing environmental conditions require regulated transcription Regulation of transcription includes both initiation and amount Involves regulatory transcription factors that bind to DNA in the vicinity of a promoter and affect transcription of one or more nearby genes 11 Trans-acting Factors Interact with Cis-regulatory Elements to Regulate Gene Expression Cis-regulatory elements (cis-acting) Cis = same side as Regions of non-coding DNA sequence which regulate the transcription of neighboring genes Examples: promoters, enhancers, silencers (DNA sequences) Trans-regulatory elements (trans-acting) Trans = across from DNA sequences encoding upstream regulators (i.e., trans- acting factors), which may modify or regulate the expression of distant genes Examples: transcription factors, repressors, activators (proteins) Negative & Positive Control of Transcription Negative control of transcription involves a repressor protein binding to a regulatory DNA sequence and inhibiting transcription of a gene Positive control of transcription involves an activator protein binding to regulatory DNA sequence and initiating transcription of a gene 13 Repressor Proteins – Negative Control Repressor proteins are regulatory proteins that exert negative control of transcription They can be activated or inactivated via interactions with other compounds Activated repressors bind to regulatory sequences such as operators, blocking transcription initiation Repressor proteins usually have two active sites: The DNA-binding domain locates and binds operator or other target DNA sequences The allosteric domain binds a molecule or protein, which causes a conformational change in the DNA-binding domain 14 Allosteric Changes Affect Repressor Function Some repressor proteins undergo inactivation of their DNA-binding domains due to allosteric changes caused by binding an inducer at the allosteric site Other repressors undergo activation of the DNA-binding domain upon allosteric binding to a corepressor 15 Activator Proteins – Positive Control Positive control of transcription is accomplished by activator proteins that bind regulatory DNA sequences called activator binding sites Activator proteins have a DNA-binding domain and a allosteric domain Activator protein binding facilitates RNA polymerase binding at promoters and helps initiate transcription 16 Two Modes of Action for Positive Control In one mode, the DNA- binding domain is inactive until an effector binds the allosteric domain and causes a conformational change that activates the DNA-binding domain In another mode, certain activator proteins have a DNA-binding domain that is converted to an inactive conformation by an inhibitor binding to the allosteric domain 17 The lac Operon is an Inducible System under Negative and Positive Control In bacteria, clusters of genes undergoing coordinated transcriptional regulation by a shared regulatory region are called operons Transcribed as polycistronic mRNA Genes in an operon nearly always participate in the same metabolic or biosynthetic pathway The lactose (lac) operon of E. coli produces three polypeptides for metabolizing lactose 18 Lactose Metabolism The monosaccharide, glucose, is the preferred energy source of E. coli It is metabolized by the biochemical pathway glycolysis Lactose, a disaccharide, can serve as an alternate carbon source; its use is controlled by the lac operon, which is an inducible operon system Inducible systems are turned on only when an inducer compound is present 19 Lactose Utilization lac+ E. coli produce a gated channel in the cell membrane that allows lactose to enter the cell and the enzyme b- galactosidase to break the b-galactoside linkage lac E. coli cannot use lactose Glucose produced by lactose breakdown enters glycolysis; galactose is processed to produce glucose, which then enters glycolysis The breakdown also produces a small amount of allolactose, which acts as an inducer molecule 20 Lac Operon Structure The lac operon consists of a multipart regulatory region and three structural genes The regulatory region contains: 1. the promoter (lacP) that binds RNA polymerase, 2. the operator (lacO) that binds the lac repressor protein 3. the CAP-cAMP region (CAP site) E.coli chromosomal DNA lac repressor gene lac operon lacI lacZ lacY lacA Encodes lac Encodes β-galactosidase Encodes lactose Encodes i promoter repressor CAP site Operator permease galactoside lac (lacO) transacetylase terminator lac promoter (lacP) 21 Lac Operon Regulatory Region 22 Lac Operon Structural Genes The three structural genes are: 1. lacZ, which encodes b-galactosidase 2. lacY, which encodes permease 3. lacA, which encodes transacetylase Transcribed as a single, polycistronic mRNA, which is translated to produce the three polypeptides E.coli chromosomal DNA lac repressor gene lac operon lacI lacZ lacY lacA Encodes lac Encodes β-galactosidase Encodes lactose Encodes i promoter repressor CAP site Operator permease galactoside lac (lacO) Transcription transacetylase terminator lac promoter la (lacP) cY lacZ lacA Polycistronic mRNA 23 LacI lacI gene is next to the lac operon, but not part of it lacI encodes the lac repressor protein, which is constitutively expressed The lac repressor protein has a DNA-binding domain that binds to the lacO sequence and an allosteric domain that binds the inducer, allolactose E.coli chromosomal DNA lac repressor gene lac operon lacI lacZ lacY lacA Encodes lac Encodes β-galactosidase Encodes lactose Encodes i promoter repressor CAP site Operator permease galactoside lac (lacO) transacetylase terminator lac promoter (lacP) 24 Lac Operon Function The lac operon is transcriptionally silent when glucose is available or lactose is absent When there is no allolactose in the cell, the lac repressor protein binds to lacO, preventing transcription This is an example of negative control lac regulatory gene lac operon RNA polymerase lac promoter Operator lacI lacP lacO lacZ lacY lacA mRNA Lac repressor binds to the operator and inhibits transcription. 25 Lac repressor (active) Lac Operon Function cont. When glucose is not available and lactose is present, transcription of the lac operon is induced Allolactose binds to the allosteric domain of the lac repressor altering the DNA-binding domain and preventing binding to the operator RNA polymerase lacI lacP lacO lacZ lacY lacA Transcription Polycistronic mRNA mRNA Translation β-Galactosidase Lactose Galactoside permease transacetylase Allolactose Conformational change The binding of allolactose to the lac repressor causes a conformational change that prevents the lac repressor from binding to the operator site. Lac repressor (inactive) 26 Levels of lac Operon Transcription The lac repressor protein binds reversibly to the operator sequence and is occasionally released from lacO, leading to a very low level of transcription, even in the absence of lactose A small amount of permease and b-galactosidase proteins allows for import of lactose into a cell and production of allolactose to bind the lac repressor 27 Additional Control of the lac Operon The inducer-repressor complex alone is not sufficient to generate enough copies of the lac operon mRNA for metabolism of lactose Positive control of the lac operon occurs at the CAP binding site of the lac promoter The site attracts the CAP-cAMP complex composed of the catabolic activator protein (CAP) and cyclic AMP (cAMP) cAMP (cyclic adenosine monophosphate) is synthesized from ATP (adenosine triphosphate) by adenylate cyclase 28 Positive Control of the lac Operon When glucose is present, little cAMP is produced and the CAP-cAMP complex cannot form Without CAP-cAMP bound to the lac promoter, lac gene transcription is very inefficient; this is called catabolite repression CAP site When glucose is absent, cAMP levels increase, Three- resulting in the formation of dimensional structure of CAP bound DNA the CAP–cAMP complex, to the CAP site cAMP which binds to the CAP site CAP dimer of the promoter, stimulating CAP site transcription Promoter Operator CAP cAMP Transcription occurs Binding of RNA polymerase to promoter is enhanced 29 mRNA by CAP binding. RNA polymerase Positive Control of the lac Operon 30 Dual Control of the lac Operon Allolactose high, cAMP low Low rate of CAP site Promoter Operator transcription Allolactose CAP Transcription is Lac repressor low due to a lack (Inactive) (inactive) of CAP binding. (a) Lactose high, glucose high When both lactose and glucose are high, the lac operon is shut off Glucose uptake causes cAMP levels to drop CAP does not activate transcription Bacterium uses glucose 31 Dual Control of the lac Operon CAP site Allolactose high, cAMP high High rate of Promoter Operator transcription CAP cAMP Binding of RNA polymerase Allolactose to promoter is enhanced by Lac repressor CAP binding. Transcription (inactive) rate is high. (b) Lactose high, glucose low When lactose is high and glucose is low, the lac operon is turned on Allolactose levels rise and prevent lac repressor from binding to operator CAP is bound to the CAP site Bacterium uses lactose 32 Dual Control of the lac Operon CAP site Promoter Operator CAP site Promoter Operator Allolactose low, Allolactose low, cAMP low cAMP high Very low rate of Very low rate of transcription transcription CAP cAMP Transcription is inhibited by lack of RNA polymerase binds, but CAP CAP binding and by the binding of transcription is blocked by the lac repressor. the binding of the lac repressor. (Inactive) (c) Lactose low, glucose high (d) Lactose low, glucose low When lactose is low, the lac operon is shut off regardless if glucose is high or low Under low lactose conditions, lac repressor prevents transcription of lac operon 33 Mutations in lac Operon Mutations in lac Operon Mutations in lac Operon Mutations in lac Operon Mutations in lac Operon Mutations in lac Operon The lac Operon https://www.youtube.com/watch?v=oBwtxdI1zvk E.coli chromosomal DNA lac repressor gene lac operon lacI lacZ lacY lacA Encodes lac Encodes β-galactosidase Encodes lactose Encodes i promoter repressor CAP site Operator permease galactoside lac (lacO) Transcription transacetylase terminator lac promoter la (lacP) cY lacZ lacA Polycistronic mRNA 40 Regulation of Metabolic Pathways Chemical reactions occur in metabolic pathways with each step coordinated by a specific enzyme Catabolic pathways breakdown cellular components Anabolic pathways synthesize cellular components Operons involved in anabolic pathways operate via the end product acting to block transcription These are called repressible operons Certain repressible operons have a second regulatory capability called attenuation, which can fine-tune transcription to match the immediate needs of the cell 41 The trp Operon In E. coli, the trp operon encodes enzymes required to make the amino acid tryptophan The trp operon contains five structural genes and a regulatory region with a promoter (trpP), operator (trpO), and leader region (trpL) that contains the attenuator region Protein products of the structural genes (trpE, trpD, trpC, trpB, and trpA) are responsible for tryptophan synthesis A sixth gene outside the operon encodes trpR, a repressor protein that is activated when bound to tryptophan 42 43 Feedback Inhibition of the trp Operon When present, tryptophan (a) Tryptophan present acts as a corepressor by trpE trpD trpC trpB trpA binding to the trp trpR P O repressor protein and activating it An activated repressor Tryptophan then binds to trpO and prevents transcription When tryptophan is (b) Tryptophan absent absent, the trp repressor protein cannot bind the RNA polymerase operator, which allows trpR transcription P O As more tryptophan is trpE trpD trpC trpB trpA made, the operon is Trp repressor Transcription increasingly repressed mRNA 44 Regulation of Metabolic Pathways lac repressor binds to its operator in the absence of its small effector molecule Inducible – allolactose induces transcription Operons for catabolism are often inducible Genes turned off unless appropriate substance available trp repressor binds to its operator only in the presence of its small effector molecule Repressible – tryptophan represses transcription Operons for anabolism are often repressible When enough of product present, genes are turned off to prevent overproduction 45 X