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CAMPBELL BIOLOGY TENTH EDITION Reece • Urry • Cain • Wasserman • Minorsky • Jackson Operons and pGLO pre lab © 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 © 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 © 2014 Pearson Education, Inc. Figure 18.2 Precursor Feedback inhibition trpE Enzyme 1 trpD Regulation of gene expression Enzyme 2 trpC trpB Enzyme 3 trpA Tryptophan (a) Regulation of enzyme activity © 2014 Pearson Education, Inc. (b) Regulation of enzyme production 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 © 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 © 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. © 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 © 2014 Pearson Education, Inc. Figure 18.3 trp operon DNA Promoter Regulatory gene Promoter Genes of operon trpE trpR RNA polymerase Operator Start codon trpD trpC trpB trpA Stop codon 3 mRNA 5 mRNA 5 Inactive repressor Protein E DNA trpE No RNA made 3 mRNA 5 Protein Active repressor Tryptophan (corepressor) (b) Tryptophan present, repressor active, operon off © 2014 Pearson Education, Inc. C B Polypeptide subunits that make up enzymes for tryptophan synthesis (a) Tryptophan absent, repressor inactive, operon on trpR D A 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 © 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 © 2014 Pearson Education, Inc. Figure 18.4a Regulatory gene Promoter Operator DNA lac I IacZ No RNA made 3′ mRNA 5′ Protein RNA polymerase Active repressor (a) Lactose absent, repressor active, operon off © 2014 Pearson Education, Inc. Figure 18.4b lac operon DNA lac I lacZ RNA polymerase mRNA 3′ Start codon lacY lacA Stop codon mRNA 5′ 5′ Protein β-Galactosidase Inactive repressor Allolactose (inducer) (b) Lactose present, repressor inactive, operon on © 2014 Pearson Education, Inc. Permease Transacetylase 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 © 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 © 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 © 2014 Pearson Education, Inc. Figure 18.5a Promoter Operator DNA lac I lacZ CAP-binding site cAMP Active CAP Inactive CAP Allolactose RNA polymerase binds and transcribes Inactive lac repressor (a) Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized © 2014 Pearson Education, Inc. Figure 18.5b Promoter DNA lac I CAP-binding site Inactive CAP lacZ Operator RNA polymerase less likely to bind Inactive lac repressor (b) Lactose present, glucose present (cAMP level low): little lac mRNA synthesized © 2014 Pearson Education, Inc. pGLO lab © 2014 Pearson Education, Inc. Using GFP as a biological tracer http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html © 2014 Pearson Education, Inc. With permission from Marc Zimmer Transformation Procedure Overview Day 1 Day 2 © 2014 Pearson Education, Inc. What is Transformation? GFP • Uptake of foreign DNA, often a circular plasmid Beta-lactamase Ampicillin Resistance © 2014 Pearson Education, Inc. Gene Expression • Beta Lactamase – Ampicillin resistance • Green Fluorescent Protein (GFP) – Aequorea victoria jellyfish gene • araC regulator protein – Regulates GFP transcription © 2014 Pearson Education, Inc. Bacterial Transformation Cell wall GFP Bacterial chromosomal DNA Beta lactamase (ampicillin resistance) pGLO plasmids © 2014 Pearson Education, Inc. Transcriptional Regulation • Lactose operon • Arabinose operon • pGLO plasmid © 2014 Pearson Education, Inc. Transcriptional Regulation ara Operon lac Operon LacI Z Y A ara C Z Y A araC © 2014 Pearson Education, Inc. Y A B A D RNA Polymerase RNA Polymerase Z A D Effector (Arabinose) Effector (Lactose) LacI B araC B A D Gene Regulation ara GFP Operon ara Operon ara C B A D araC Effector (Arabinose) Effector (Arabinose) araC B A D araC RNA Polymerase araC B A D © 2014 Pearson Education, Inc. GFP Gene GFP Gene RNA Polymerase araC GFP Gene