Anticipatory Questions,1. What might happen if an organism had its cells expressing all genes within the genome all the time? 2. At what levels can control of cellular activities/pathways be controlled? 3. Based on our discussions up to this point, what do you think the term “negative feedback” means? 4. What steps are involved in the initiation of prokaryotic transcription?,Learning Objectives,understand that regulation of gene expression is a means by which to control timing and rate of generation regarding functional gene product (either RNA or polypeptide/protein). explain the concept of an operon in terms of components functions (promoter, operator, repressor, co-repressor, inducer, gene cluster, polycistronic transcript). compare and contrast repressible and inducible operon systems/pathways. compare and contrast negative versus positive regulation of operons apply the operon concept to gene expression as it relates to genetic engineering (specifically, our cloning and expression of the “tomato” gene).,Individual bacteria respond to environmental change by regulating their gene expression,A bacterium can tune its metabolism to the changing environment and food sources This metabolic control occurs on two levels: Adjusting activity of metabolic enzymes Regulating genes that encode metabolic enzymes,LE 18-20,Regulation of enzyme activity,Regulation of enzyme production,Enzyme 1,Regulation of gene expression,Enzyme 2,Enzyme 3,Enzyme 4,Enzyme 5,Gene 2,Gene 1,Gene 3,Gene 4,Gene 5,Tryptophan,Precursor,Feedback inhibition,Operons: The Basic Concept,In bacteria, genes are often clustered into operons, composed of An operator, an “on-off” switch A promoter Genes for metabolic enzymes An operon can be switched off by a protein called a repressor A corepressor is a small molecule that cooperates with a repressor to switch an operon off,LE 18-21a,Promoter,Promoter,DNA,trpR,Regulatory gene,RNA polymerase,mRNA,3,5,Protein,Inactive repressor,Tryptophan absent, repressor inactive, operon on,Polycistronic* mRNA,trpE,trpD,trpC,trpB,trpA,Operator,Start codon,Stop codon,trp operon,Genes of operon,E,Polypeptides that make up enzymes for tryptophan synthesis,D,C,B,A,5,* = mRNA carries the information of several genes, which are translated into several proteins,LE 18-21b_1,DNA,Protein,Tryptophan (corepressor),Tryptophan present, repressor active, operon off,mRNA,Active repressor,LE 18-21b_2,DNA,Protein,Tryptophan (corepressor),Tryptophan present, repressor active, operon off,mRNA,Active repressor,No RNA made,Trp Operon Animation,http:/bcs.whfreeman.com/thelifewire/content/chp13/1302002.html,Repressible and Inducible Operons: Two Types of Negative Gene Regulation,A repressible operon is one that is usually on; binding of a repressor to the operator shuts off transcription The trp operon is a repressible operon An inducible operon is one that is usually off; a molecule called an inducer inactivates the repressor and turns on transcription The classic example of an inducible operon is the lac operon, which contains genes coding for enzymes in hydrolysis and metabolism of lactose,LE 18-22a,DNA,lacl,Regulatory gene,mRNA,5,3,RNA polymerase,Protein,Active repressor,No RNA made,lacZ,Promoter,Operator,Lactose absent, repressor active, operon off,LE 18-22b,DNA,lacl,mRNA,5,3,lac operon,Lactose present, repressor inactive, operon on,lacZ,lacY,lacA,RNA polymerase,Polycistronic mRNA,Protein,Allolactose (inducer),Inactive repressor,-Galactosidase,Permease,Transacetylase,5,Lac Operon Animation,http:/www.sumanasinc.com/webcontent/animations/content/lacoperon.html,Inducible enzymes usually function in catabolic pathways Repressible enzymes usually function in anabolic pathways Regulation of the trp and lac operons involves negative control of genes because operons are switched off by the active form of the repressor,Positive Gene Regulation,Some operons are also subject to positive control through a stimulatory activator protein, such as catabolite activator protein (CAP) When glucose (a preferred food source of E. coli ) is scarce, the lac operon is activated by the binding of CAP When glucose levels increase, CAP detaches from the lac operon, turning it off,LE 18-23a,DNA,cAMP,lacl,CAP-binding site,Promoter,Active CAP,Inactive CAP,RNA polymerase can bind and transcribe,Operator,lacZ,Inactive lac repressor,Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized,LE 18-23b,DNA,lacl,CAP-binding site,Promoter,RNA polymerase cant bind efficiently,Operator,lacZ,Inactive lac repressor,Inactive CAP,Lactose present, glucose present (cAMP level low): little lac mRNA synthesized,Catabolite Activator Protein Mechanism,http:/highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter18/animations.html# Click on “combination of switches - the lac operon”,The Arabinose Operon - A Composite of Negative & Positive Regulation,a) In the presence of arabinose: CAP-cAMP complex and araC-arabino