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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.
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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 TRANSCRIPTION LE 17-3-2 mRNA Ribosome TRANSLATION Polypeptide Prokaryotic cell
transcription of DNA to mRNA starts at the promoter, ends at the terminator
Some important prerequisite facts • DNA is the code to make a protein • Some proteins are attracted to specific sequences of DNA • Affinity for DNA sequences can change with changes in protein conformation • A special protein (RNA polymerase) transcribes DNA RNA • 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, 632-642 (August 2004)
- galactosidase b 10
b - galactosidase H O 2 galactose lactose b - galactosidase glucose (aka lactase in humans) 11
b - galactosidase Regulation b Why Regulate - galactosidase ? • b Levels at which - galactosidase can be • regulated: Genetic • Biochemical • 12
Regulation of enzyme production Regulation of enzyme activity The manufacture of enzymes responsible for the biosynthesis of the amino acid tryptophan is also closely regulated Precursor Feedback inhibition Enzyme 1 Gene 1 LE 18-20 Gene 2 Enzyme 2 Regulation of gene expression Gene 3 Enzyme 3 Enzyme 4 Gene 4 Gene 5 Enzyme 5 Tryptophan
trp operon Promoter Promoter Prokaryotic Operon structure ensures efficient regulation of transcription Genes of operon DNA trpB trpA trpE trpC trpD trpR Operator Stop codon RNA polymerase Regulatory gene Start codon 3¢ mRNA 5¢ mRNA 5¢ D B E C A Inactive repressor Protein 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
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
DNA LE 18-21b_1 mRNA Active repressor Protein Tryptophan (corepressor) Tryptophan present, repressor active, operon off
DNA No RNA made LE 18-21b_2 mRNA Active repressor Protein Tryptophan (corepressor) Tryptophan present, repressor active, operon off
Repressor Protein Repressor mRNA Promoter Operator Structural Genes RNA Polymerase Regulator Gene Basic Operon Regulation NO TRANSCRIPTION
Tryptophan Operon Tryptophan Present Regulator Gene Promoter Operator Attenuator Structural Genes RNA Polymerase NO TRANSCRIPTION trpRmRNA 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 trpRmRNA TrpR protein (homodimer) TrpR aporepressor (cannot bind to operator)
Tryptophan Repressor Protein TrpR protein subunits Tryptophan (co-repressor) 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
Promoter Regulatory gene Operator lacl lacZ DNA LE 18-22a No RNA made 3¢ mRNA RNA polymerase 5¢ Active repressor Protein Lactose absent, repressor active, operon off
lac operon DNA lacl lacY lacA lacZ LE 18-22b RNA polymerase 3¢ mRNA mRNA 5¢ 5¢ Transacetylase Permease -Galactosidase Protein Inactive repressor Allolactose (inducer) Lactose present, repressor inactive, operon on
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
Promoter DNA lacl lacZ LE 18-23a 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
Promoter DNA lacl lacZ LE 18-23b CAP-binding site Operator RNA polymerase can’t bind Inactive CAP Inactive lac repressor Lactose present, glucose present (cAMP level low): little lac mRNA synthesized
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.
mRNA β-gal Operons can be cut and pasted together to make operon fusions Tryptophan Operon Lactose Operon Repressor lacI T Pro. Oper. Z gene Y gene A gene Pro. Oper. TrpE, D, C, B, A Att. Operator Promoter Z gene Y gene A gene mutant trpR-containing plasmid
mRNA β-gal If the repressor is knocked out, what will happen in the presence of Tryptophan? Tryptophan Operon Lactose Operon Repressor lacI T Pro. Oper. Z gene Y gene A gene Pro. Oper. TrpE, D, C, B, A Att. Operator Promoter Z gene Y gene A gene
mRNA β-gal What if we add a plasmid which contains the TrpR gene? With tryptophan? Without? Tryptophan Operon Lactose Operon Repressor lacI T Pro. Oper. Z gene Y gene A gene Pro. Oper. TrpE, D, C, B, A Att. Operator Promoter Z gene Y gene A gene mutant trpR-containing plasmid
Another engineered plasmid with fusion Operon: pGLO araC ori pGLO GFP bla • Manufactured by a private corporation • AraC- arabinose gene • GFP- Green Fluorescent protein • bla- Beta-lactamase • ori- you know this… VandePol
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”?
Which colonies will glow? LB/Amp LB/Amp/Ara LB 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.
DNA polymerase binds to the ori Parental (template) strand 0.25 µm Origin of replication Daughter (new) strand LE 16-12 Replication fork Bubble Two daughter DNA molecules In this micrograph, three replication bubbles are visible along the DNA of a cultured Chinese hamster cell (TEM). In eukaryotes, DNA replication begins at may sites along the giant DNA molecule of each chromosome.
ara GFP Operon ara Operon araC GFP Gene araC B A D Effector(Arabinose) Effector (Arabinose) araC B A D araC GFP Gene RNA Polymerase RNA Polymerase B A D araC araC GFP Gene Gene Regulation 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 GFP can be fused to cellular proteins http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html With permission from Marc Zimmer
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