1. CAMPBELL BIOLOGY Reece Urry Cain Wasserman Minorsky Jackson © 2014 Pearson Education, Inc. TENTH EDITION CAMPBELL BIOLOGY Reece Urry Cain Wasserman Minorsky Jackson TENTH EDITION Operons and pGLO pre lab

2. © 2014 Pearson Education, Inc. Differential Expression of Genes  Prokaryotes and eukaryotes precisely regulate gene expression in response to environmental conditions  In multicellular eukaryotes, gene expression regulates development and is responsible for differences in cell types  RNA molecules play many roles in regulating gene expression in eukaryotes

3. © 2014 Pearson Education, Inc. Concept 18.1: Bacteria often respond to environmental change by regulating transcription  Natural selection has favored bacteria that produce only the products needed by that cell  A cell can regulate the production of enzymes by feedback inhibition or by gene regulation  One mechanism for control of gene expression in bacteria is the operon model

4. © 2014 Pearson Education, Inc. Figure 18.2 Precursor Feedback inhibition Tryptophan (b) Regulation of enzyme production (a) Regulation of enzyme activity Regulation of gene expression trpE trpD trpC trpB trpA Enzyme 1 Enzyme 2 Enzyme 3

5. © 2014 Pearson Education, Inc. Operons: The Basic Concept  A cluster of functionally related genes can be coordinately controlled by a single “on-off switch”  The “switch” is a segment of DNA called an operator usually positioned within the promoter  An operon is the entire stretch of DNA that includes the operator, the promoter, and the genes that they control

6. © 2014 Pearson Education, Inc.  The operon can be switched off by a protein repressor  The repressor prevents gene transcription by binding to the operator and blocking RNA polymerase  The repressor is the product of a separate regulatory gene  Repressors are often made continuously by the cell, at low levels

7. © 2014 Pearson Education, Inc.  The repressor can be in an active or inactive form, depending on the presence of other molecules  A corepressor is a molecule that cooperates with a repressor protein to switch an operon off  A corepressor often binds allosterically to the repressor to change its shape and allow it to bind to the operator.

8. © 2014 Pearson Education, Inc. E. coli example  For example, E. coli can synthesize the amino acid tryptophan when it has insufficient tryptophan  By default the trp operon is on and the genes for tryptophan synthesis are transcribed  When tryptophan is present, it binds to the trp repressor protein, which turns the operon off  The repressor is active only in the presence of its corepressor tryptophan; thus the trp operon is turned off (repressed) if tryptophan levels are high

9. © 2014 Pearson Education, Inc. Figure 18.3 Promoter DNA trpR Regulatory gene RNA polymerase mRNA 55 33 Protein Inactive repressor mRNA 5  (a) Tryptophan absent, repressor inactive, operon on DNA mRNA Protein Active repressor No RNA made Promoter trp operon Genes of operon trpE trpDtrpCtrpBtrpA Operator Start codon Stop codon trpRtrpE Tryptophan (corepressor) (b) Tryptophan present, repressor active, operon off 33 55 Polypeptide subunits that make up enzymes for tryptophan synthesis EDCB A

10. © 2014 Pearson Education, Inc. 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

11. © 2014 Pearson Education, Inc.  The lac operon is an inducible operon and contains genes that code for enzymes used in the hydrolysis and metabolism of lactose  By itself, the lac repressor is active and switches the lac operon off  A molecule called an inducer inactivates the repressor to turn the lac operon on

12. © 2014 Pearson Education, Inc. Figure 18.4a Promoter DNA Regulatory gene mRNA 5′5′ 3′3′ Operator RNA polymerase Active repressor No RNA made IacZ (a) Lactose absent, repressor active, operon off Protein lac I

13. © 2014 Pearson Education, Inc. Figure 18.4b lacZ lacY lacA (b) Lactose present, repressor inactive, operon on 5′5′ DNA RNA polymerase mRNA 3′3′ Protein Inactive repressor Allolactose (inducer) mRNA 5′ lac I Start codonStop codon Permease Transacetylase β-Galactosidase lac operon

14. © 2014 Pearson Education, Inc.  Inducible enzymes usually function in catabolic pathways; their synthesis is induced by a chemical signal  Repressible enzymes usually function in anabolic pathways; their synthesis is repressed by high levels of the end product  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. © 2014 Pearson Education, Inc. Positive Gene Regulation  Some operons are also subject to positive control through a stimulatory protein, such as catabolite activator protein (CAP), an activator of transcription  When glucose (a preferred food source of E. coli) is scarce, CAP is activated by binding with cyclic AMP (cAMP)  Activated CAP attaches to the promoter of the lac operon and increases the affinity of RNA polymerase, thus accelerating transcription

16. © 2014 Pearson Education, Inc.  When glucose levels increase, CAP detaches from the lac operon, and transcription returns to a normal rate  CAP helps regulate other operons that encode enzymes used in catabolic pathways

17. © 2014 Pearson Education, Inc. Figure 18.5a DNA Promoter Operator CAP-binding site cAMP Active CAP Inactive CAP RNA polymerase binds and transcribes lac I Allolactose Inactive lac repressor (a) Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized lacZ

18. © 2014 Pearson Education, Inc. Figure 18.5b Promoter DNA lacZ CAP-binding site RNA polymerase less likely to bind Operator Inactive CAP Inactive lac repressor (b) Lactose present, glucose present (cAMP level low): little lac mRNA synthesized lac I

19. © 2014 Pearson Education, Inc. pGLO lab

20. © 2014 Pearson Education, Inc. Using GFP as a biological tracer http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html With permission from Marc Zimmer

21. © 2014 Pearson Education, Inc. Transformation Procedure Overview Day 1 Day 2

22. © 2014 Pearson Education, Inc. What is Transformation? Uptake of foreign DNA, often a circular plasmid GFP Beta-lactamase Ampicillin Resistance

23. © 2014 Pearson Education, Inc. Gene Expression Beta Lactamase –Ampicillin resistance Green Fluorescent Protein (GFP) –Aequorea victoria jellyfish gene araC regulator protein –Regulates GFP transcription

24. © 2014 Pearson Education, Inc. Bacterial Transformation Beta lactamase (ampicillin resistance) pGLO plasmids Bacterial chromosomal DNA Cell wall GFP

25. © 2014 Pearson Education, Inc. Transcriptional Regulation Lactose operon Arabinose operon pGLO plasmid

26. © 2014 Pearson Education, Inc. Transcriptional Regulation BAD araC BAD RNA Polymerase Effector (Arabinose) araC BAD ara Operon RNA Polymerase ZYA ZYA LacI Effector (Lactose) ZYA LacI lac Operon

27. © 2014 Pearson Education, Inc. Gene Regulation RNA Polymerase araC ara GFP Operon GFP Gene araC GFP Gene araC GFP Gene Effector (Arabinose) BAD araC BAD RNA Polymerase Effector (Arabinose) araC BAD ara Operon