Anticipatory Questions

1. 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?

2. 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).

3. 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

4. LE 18-20 Regulation of enzyme production Regulation of enzyme activity Precursor Feedback inhibition Enzyme 1 Gene 1 Gene 2 Enzyme 2 Regulation of gene expression Gene 3 Enzyme 3 Enzyme 4 Gene 4 Gene 5 Enzyme 5 Tryptophan

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

6. LE 18-21a trp operon Promoter Promoter Genes of operon DNA trpB trpA trpE trpC trpD trpR Operator Stop codon RNA polymerase Regulatory gene Start codon 3¢ 5¢ mRNA 5¢ Polycistronic* mRNA D B E C A Inactive repressor Protein 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

7. LE 18-21b_1 DNA mRNA Active repressor Protein Tryptophan (corepressor) Tryptophan present, repressor active, operon off

8. LE 18-21b_2 DNA No RNA made mRNA Active repressor Protein Tryptophan (corepressor) Tryptophan present, repressor active, operon off

9. Trp Operon Animation • http://bcs.whfreeman.com/thelifewire/content/chp13/1302002.html

10. 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

11. LE 18-22a Promoter Regulatory gene Operator lacl lacZ DNA No RNA made 3¢ mRNA RNA polymerase 5¢ Active repressor Protein Lactose absent, repressor active, operon off

12. LE 18-22b lac operon DNA lacl lacY lacA lacZ RNA polymerase 3¢ mRNA 5¢ 5¢ Polycistronic mRNA Transacetylase Permease -Galactosidase Protein Inactive repressor Allolactose (inducer) Lactose present, repressor inactive, operon on

13. Lac Operon Animation http://www.sumanasinc.com/webcontent/animations/content/lacoperon.html

14. 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

15. 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

16. LE 18-23a Promoter DNA lacl lacZ RNA polymerase can bind and transcribe Operator CAP-binding site Active CAP cAMP Inactive lac repressor Inactive CAP Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized

17. LE 18-23b Promoter DNA lacl lacZ CAP-binding site Operator RNA polymerase can’t bind efficiently Inactive CAP Inactive lac repressor Lactose present, glucose present (cAMP level low): little lac mRNA synthesized

18. Catabolite Activator Protein Mechanism • http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter18/animations.html# • Click on “combination of switches - the lac operon”

19. The Arabinose Operon - A Composite of Negative & Positive Regulation • a) In the presence of arabinose: • CAP-cAMP complex and araC-arabinose complex bind to initiator region • this allows RNA polymerase to bind to the promoter • transcription begins • 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

20. Application of Operons: Regulatory gene Promoter for the cluster of genes B, A, and D Operator (part of the promoter)

21. Arabinose operon with in-frame foreign DNA inserted: araC regulatory gene Gene B Gene A Tomato gene Gene D Gene D Tomato gene repressor transcription Inducer (arabinose) stop start stop stop start start 3´ stop start translation Polycistronic mRNA 5´ translation translation translation Red Fluorescent Protein (RFP) Protein B Protein A Protein D