Transcript video slide
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? Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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). Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 18-20 Regulation of enzyme activity Precursor Regulation of enzyme production Feedback inhibition Enzyme 1 Gene 1 Enzyme 2 Gene 2 Regulation of gene expression Enzyme 3 Gene 3 Enzyme 4 Gene 4 Enzyme 5 Tryptophan Gene 5 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 18-21a trp operon Promoter Promoter Genes of operon DNA trpE trpR trpD trpC trpB trpA C B A Operator Regulatory gene mRNA Protein 3 RNA polymerase 5 Start codon Stop codon 5 Polycistronic* mRNA Inactive repressor E D Polypeptides that make up enzymes for tryptophan synthesis Tryptophan absent, repressor inactive, operon on * = mRNA carries the information of several genes, which are translated into several proteins LE 18-21b_1 DNA mRNA Active repressor Protein Tryptophan (corepressor) Tryptophan present, repressor active, operon off LE 18-21b_2 DNA No RNA made mRNA Active repressor Protein Tryptophan (corepressor) Tryptophan present, repressor active, operon off Trp Operon Animation • http://bcs.whfreeman.com/thelifewire/content/chp1 3/1302002.html Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 18-22a Promoter Regulatory gene Operator lacl DNA lacZ No RNA made 3 mRNA 5 Protein RNA polymerase Active repressor Lactose absent, repressor active, operon off LE 18-22b lac operon DNA lacZ lacl 3 lacY lacA Permease Transacetylase RNA polymerase mRNA 5 5 Polycistronic mRNA -Galactosidase Protein Allolactose (inducer) Inactive repressor Lactose present, repressor inactive, operon on Lac Operon Animation http://www.sumanasinc.com/webcontent/animations/ content/lacoperon.html Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings • 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 18-23a Promoter DNA lacl lacZ CAP-binding site Active CAP cAMP Inactive CAP RNA Operator polymerase can bind and transcribe Inactive lac repressor Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized LE 18-23b Promoter DNA lacl CAP-binding site Inactive CAP lacZ Operator RNA polymerase can’t bind efficiently Inactive lac repressor Lactose present, glucose present (cAMP level low): little lac mRNA synthesized Catabolite Activator Protein Mechanism • http://highered.mcgrawhill.com/sites/0072437316/student_view0/chapter1 8/animations.html# • Click on “combination of switches - the lac operon” Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings The Arabinose Operon - A Composite of Negative & Positive Regulation a) In the presence of arabinose: •CAP-cAMP complex and araCarabinose complex bind to initiator region •this allows RNA polymerase to bind to the promoter •transcription begins Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings b) In the absence of arabinose: •araC protein assumes a different conformation •acts as a repressor •binds to araI and a second operator region araO •forms a loop •this loop prevents transcription Application of Operons: Regulatory gene Promoter for the cluster of genes B, A, and D Operator (part of the promoter) Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Arabinose operon with in-frame foreign DNA inserted: araC regulatory gene Gene B Gene A Tomato gene Tomato gene Gene Gene DD repressor transcription Inducer (arabinose) start Polycistronic mRNA 5´ stop start translation Protein B stop start Protein A Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 3´ stop start translation stop translation translation Red Fluorescent Protein (RFP) Protein D