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Bio 101A GE III results day Which one of the following occurs when RNA polymerase attaches to the promoter DNA? A) elongation of the growing RNA molecule B) termination of the RNA molecule C) addition of nucleotides to the DNA template D) initiation of a new RNA molecule E) initiation of a new polypeptide chain Which of the following is the enzyme that HIV uses to synthesize DNA on an RNA template? A) ligase B) RNA polymerase C) terminator enzyme D) reverse transcriptase E) None of the choices are correct. Prokaryotic Gene Regulation Bio 101A • Operon structure and function • Positive/Negative gene regulation • Promoters, operators, repressors, inducers Q: What is “regulation”? "When I was warning about the danger ahead on Wall Street months ago because of the lack of oversight, Senator McCain was telling the Wall Street Journal -- and I quote -- 'I'm always for less regulation.' " – Sen. Barack Obama “Senator Obama was silent on the regulation of Fannie Mae and Freddie Mac, and his Democratic allies in Congress opposed every effort to rein them in…last year he said that subprime loans had been, quote, “a good idea.””- Sen. John McCain Enzymes are coded for by genes • DNA is the code to make proteins • Enzymes are made of protein • In order for a cell to make an enzyme, it must access the DNA for that enzyme • Enzymes are very specific to their task V. fischeri interacts symbiotically with a squid • Helps the squid camouflage itself during nocturnal hunting • 95% of colonies are expelled daily • The rest are fed in pouches in the squid’s tissue • Bacterium has an interest in regulating expression of luciferase gene V. fischeri interacts symbiotically with a squid The winnowing: establishing the squid–vibrio symbiosis Spencer V. Nyholm & Margaret McFall-Ngai Nature Reviews Microbiology 2, 632-642 (August 2004) Enzyme Regulation maintains Efficiency • Enzymes: General Background • b-galactosidase • Gene Regulation • Biochemical Regulation • Experiment 9 b-galactosidase 10 b-galactosidase H 2O galactose lactose b-galactosidase (aka lactase in humans) glucose 11 b-galactosidase Regulation • Why Regulate b-galactosidase • Levels at which regulated: b-galactosidase ? can be • Genetic • Biochemical 12 The lac operon regulates when βgalactosidase is made When lactose is present, transcription is activated 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 • An operon is a collection of prokaryotic genes transcribed together on a single mRNA transcript to serve a single purpose • Composed of – An operator, an “on-off” switch – A promoter – Genes for metabolic enzymes • Can be switched off by a repressor protein • A corepressor is a small molecule that binds to a repressor to switch an operon off Prokaryotic Operon structure ensures efficient regulation of transcription trp operon Promoter Promoter Genes of operon DNA Regulatory gene mRNA Protein trpE trpR 3 RNA polymerase trpD trpC trpB trpA C B A Operator Start codon Stop codon mRNA 5 5 E Inactive repressor D Polypeptides that make up enzymes for tryptophan synthesis Tryptophan absent, repressor inactive, operon on The tryptophan biosynthesis operon is repressible by the presence of its product, tryptophan 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 Basic Operon Regulation Repressor Protein NO TRANSCRIPTION RNA Polymerase Repressor mRNA Regulator Gene Promoter Operator Structural Genes Tryptophan Operon Tryptophan Present Regulator Gene Promoter Operator Attenuator Structural Genes RNA Polymerase NO TRANSCRIPTION trpR mRNA Q: Why might the cell want to produce an aporepressor that is only activated by the operon’s end product? + tryptophan (corepressor) TrpR protein (homodimer) TrpR aporepressor + corepressor (can bind to operator) Tryptophan Operon Tryptophan Absent Regulator Gene Promoter Operator Attenuator Structural Genes RNA Polymerase TRANSCRIPTION trpR mRNA TrpR protein (homodimer) TrpR aporepressor (cannot bind to operator) Tryptophan Repressor Protein TrpR protein subunits Tryptophan (corepressor) DNA Repressible and Inducible Operons: Two Types of Negative Gene Regulation • A repressible operon is one that is usually on; binding of a repressor 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 LE 18-22a Promoter Regulatory gene Operator lacl DNA lacZ No RNA made 3 mRNA 5 Protein Lactose absent, repressor active, operon off RNA polymerase Active repressor LE 18-22b lac operon DNA lacZ lacl 3 mRNA 5 lacA RNA polymerase mRNA 5 b-Galactosidase Protein Allolactose (inducer) lacY Inactive repressor Lactose present, repressor inactive, operon on Permease Transacetylase • 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 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 Inactive lac repressor Lactose present, glucose present (cAMP level low): little lac mRNA synthesized In a prokaryote, a group of genes with related functions, along with their associated control sequences, defines A) an allele. B) an operon. C) a locus. D) a transposon. E) a chromosome. Operons can be cut and pasted together to make operon fusions Tryptophan Operon Repressor Pro. Oper. Att. Lactose Operon lacI TrpE, D, C, B, A Promote Operator r Z gene T Pro. Oper. Z gene Y gene Y gene A gene mRNA mutant trpRcontaining plasmid β-gal A gene If the repressor is knocked out, what will happen in the presence of Tryptophan? Tryptophan Operon Repressor Pro. Oper. Att. Lactose Operon lacI TrpE, D, C, B, A Promote Operator r Z gene T Pro. Oper. Z gene Y gene Y gene A gene mRNA β-gal A gene What if we add a plasmid which contains the TrpR gene? With tryptophan? Without? Tryptophan Operon Repressor Pro. Oper. Att. Lactose Operon lacI TrpE, D, C, B, A Promote Operator r Z gene T Pro. Oper. Z gene Y gene Y gene A gene mRNA mutant trpRcontaining plasmid β-gal A gene Another engineered plasmid with fusion Operon: pGLO Manufactured by a private corporation AraC- arabinose gene GFP- Green Fluorescent protein bla- Beta-lactamase ori- you know this… 5/23/2017 VandePol araC ori pGLO bla GFP 35 Is this: Anabolic or Catabolic? Positive or negative? Inducible or repressible? 5/23/2017 36 Expression of Green Fluorescent Protein • How do you think this fusion was made? • What are the structural sequences? The regulatory sequences? • What happens when we add arabinose sugar to these bacteria? • What do you think is meant by “reporter gene”? lac Operon and ara operon lac Operon LacI Z Y ara Operon A araC B A D Effector (Lactose) LacI Z Y A Effector (Arabinose) araC B A RNA Polymerase Z 5/23/2017 Y A D RNA Polymerase araC B A D 38 Grow? Glow? Which colonies will glow? Follow protocol On which plates will colonies grow? Which colonies will glow? 5/23/2017 39 Appendix: pGLO slides that may be helpful • Stuff about GFP, arabinose, beta-lactamase, etc. LE 16-12 DNA polymerase binds to the ori Parental (template) strand Origin of replication Bubble Daughter (new) strand 0.25 µm Replication fork Two daughter DNA molecules In eukaryotes, DNA replication begins at may sites along the giant DNA molecule of each chromosome. In this micrograph, three replication bubbles are visible along the DNA of a cultured Chinese hamster cell (TEM). On pGLO, the regulatory regions of the Arabinose operon have been glued to the structural sequences for GFP ara GFP Operon ara Operon B ara C A D araC GFP Gene Effector (Arabinose) Effector (Arabinose) Gene Regulation B A D araC araC GFP Gene RNA Polymerase RNA Polymerase araC B A D araC GFP Gene What will happen on the Ara (+) plates? What will happen on the Ara (-) plates? Green Fluorescent Protein • discovered in 1960s by Dr. Frank Johnson and colleagues • closely related to jellyfish aequorin • absorption max = 470nm • emission max = 508nm • 238 amino acids, 27kDa • “beta can” conformation: 11 antiparallel beta sheets, 4 alpha helices, and a centered chromophore • amino acid substitutions result in several variants, including YFP, BFP, and CFP 40 Å 30 Å GFP can be fused to cellular proteins Using GFP as a biological tracer http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html With permission from Marc Zimmer GFP Fusion & Protein Localization 1 Gene Fusion Control Region Gene X (no stop codon) GFP gene (in-frame with Gene X) express gene-of-interest 2 Translated Fusion Protein Protein X GFP 3 Fluorescence Visualization Fusion protein in C. elegans highlights nervous system Vesicle Transport in the Caenorhabditis elegans Nervous System SNB-1::GFP Fusion 1 Fusion of snb-1 to GFP gene Control Region For snb-1 GFP gene (in-frame with snb-1) express gene-of-interest 2 Neurotransmitter-Packaging Vesicles Present GFP-Tagged SNB-1 on Surface SynaptobrevinGFP 3 View SNB-1 (Vesicle) Distribution 2008 Nobel Prize- GFP • GFP mice The pGLO plasmid • ori- origin of replication • GFP- green fluorescent protein • bla- Beta-lactamase • araC- Arabinose • What are all the other marks? Why are they there? Beta- lactam antibiotics have a similar structure • Includes penicillin, ampicillin, and others • The beta-lactam ring is a square structure common to all Beta-lactamase can destroy a betalactam ring Breaking the ring destroys the antibiotic’s effectiveness What about araC? Arabinose is a 5-carbon sugar, different from ribose