Presentation on theme: "Prokaryotic Gene Regulation Bio 101A Operon structure and function."— Presentation transcript:
Prokaryotic Gene Regulation Bio 101A Operon structure and function
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
DNA RNA Protein Trait
LE TRANSCRIPTION DNA TRANSLATION Ribosome Polypeptide mRNA Prokaryotic cell
transcription of DNA to mRNA starts at the promoter, ends at the terminator
Some important prerequisite facts 1.DNA is the code to make a protein 2.Some proteins are attracted to specific sequences of DNA 3.Affinity for DNA sequences can change with changes in protein conformation 4.A special protein (RNA polymerase) transcribes DNA RNA 5.Regulatory sequences of DNA don’t code for any specific protein, but are still important
V. fischeri interacts symbiotically with the bobtail 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, (August 2004)
-galactosidase 11 lactose galactose glucose H 2 O -galactosidase (aka lactase in humans)
-galactosidaseRegulation 12 Why Regulate -galactosidase? Levels at which -galactosidasecan be regulated: Genetic Biochemical
LE Regulation of enzyme activity Regulation of enzyme production Enzyme 1 Regulation of gene expression Enzyme 2 Enzyme 3 Enzyme 4 Enzyme 5 Gene 2 Gene 1 Gene 3 Gene 4 Gene 5 Tryptophan Precursor Feedback inhibition The manufacture of enzymes responsible for the biosynthesis of the amino acid tryptophan is also closely regulated
Promoter DNA trpR Regulatory gene RNA polymerase mRNA 3 5 Protein Inactive repressor Tryptophan absent, repressor inactive, operon on mRNA 5 trpE trpD trpC trpBtrpA Operator Start codon Stop codon trp operon Genes of operon E Polypeptides that make up enzymes for tryptophan synthesis D C B A Prokaryotic Operon structure ensures efficient regulation of transcription The tryptophan biosynthesis operon is repressible by the presence of its product, tryptophan
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
LE 18-21b_1 DNA Protein Tryptophan (corepressor) Tryptophan present, repressor active, operon off mRNA Active repressor
LE 18-21b_2 DNA Protein Tryptophan (corepressor) Tryptophan present, repressor active, operon off mRNA Active repressor No RNA made
Basic Operon Regulation Regulator Gene PromoterOperator Structural Genes RNA Polymerase Repressor mRNA Repressor Protein NO TRANSCRIPTION
Tryptophan Operon Tryptophan Present Regulator GenePromoterOperator Structural Genes Attenuator trp R mRNA RNA Polymerase NO TRANSCRIPTION TrpR protein (homodimer) + tryptophan (corepressor) TrpR aporepressor + corepressor (can bind to operator) Q: Why might the cell want to produce an aporepressor that is only activated by the operon’s end product?
Tryptophan Operon Tryptophan Absent Regulator GenePromoterOperator Structural Genes Attenuator trp R mRNA RNA Polymerase TRANSCRIPTION TrpR protein (homodimer) TrpR aporepressor (cannot bind to operator)
Tryptophan Repressor Protein DNA Tryptophan (co- repressor) TrpR protein subunits
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 DNA lacl Regulatory gene mRNA 5 3 RNA polymerase Protein Active repressor No RNA made lacZ Promoter Operator Lactose absent, repressor active, operon off
LE 18-22b DNAlacl mRNA 5 3 lac operon Lactose present, repressor inactive, operon on lacZ lacYlacA RNA polymerase mRNA 5 Protein Allolactose (inducer) Inactive repressor -Galactosidase 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 DNA cAMP lacl CAP-binding site Promoter Active CAP Inactive CAP RNA polymerase can bind and transcribe Operator lacZ Inactive lac repressor Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized
LE 18-23b DNA lacl CAP-binding site Promoter RNA polymerase can’t bind Operator lacZ Inactive lac repressor Inactive CAP Lactose present, glucose present (cAMP level low): little lac mRNA synthesized
What about the lux operon?
Other slides I didn’t talk about The slides following show how operons can be cut and pasted together in novel ways. Regulatory sequences from one operon can be spliced to structural sequences from another, creating a whole new input/output device.
Operons can be cut and pasted together to make operon fusions lacI Repressor Oper.TrpE, D, C, B, A Att. Tryptophan OperonLactose Operon TPro.Oper. Promote r Operator Z gene Y geneA gene mRNA β-gal Pro. Z geneY geneA gene mutant trp R - containing plasmid
If the repressor is knocked out, what will happen in the presence of Tryptophan? lacI Repressor Oper.TrpE, D, C, B, A Att. Tryptophan OperonLactose Operon TPro.Oper. Promote r Operator Z gene Y geneA gene mRNA β-gal Pro. Z geneY geneA gene
What if we add a plasmid which contains the TrpR gene? With tryptophan? Without? lacI Repressor Oper.TrpE, D, C, B, A Att. Tryptophan OperonLactose Operon TPro.Oper. Promote r Operator Z gene Y geneA gene mRNA β-gal Pro. Z geneY geneA gene mutant trp R - containing plasmid
4/22/2015VandePol33 Manufactured by a private corporation AraC- arabinose gene GFP- Green Fluorescent protein bla- Beta-lactamase ori- you know this… pGLO ori bla GFP araC Another engineered plasmid with fusion Operon: pGLO
4/22/ Is this: Anabolic or Catabolic? Positive or negative? Inducible or repressible?
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”?
4/22/ Grow? Glow? Follow protocol On which plates will colonies grow? Which colonies will glow?
Appendix: pGLO slides that may be helpful Stuff about GFP, arabinose, beta-lactamase, etc.
LE In eukaryotes, DNA replication begins at may sites along the giant DNA molecule of each chromosome. Two daughter DNA molecules Parental (template) strand Daughter (new) strand 0.25 µm Replication fork Origin of replication Bubble In this micrograph, three replication bubbles are visible along the DNA of a cultured Chinese hamster cell (TEM). DNA polymerase binds to the ori
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 On pGLO, the regulatory regions of the Arabinose operon have been glued to the structural sequences for GFP What will happen on the Ara (+) plates? What will happen on the Ara (-) plates ?
Using GFP as a biological tracer With permission from Marc Zimmer GFP can be fused to cellular proteins
The pGLO plasmid ori- origin of replication GFP- green fluorescent protein bla- Beta-lactamase araC- Arabinose What are all the other marks?
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 beta- lactam ring Breaking the ring destroys the antibiotic’s effectiveness
What about araC? Arabinose is a 5-carbon sugar, different from ribose