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Prokaryotic Gene Regulation Bio 101A
Operon structure and function
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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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DNA RNA Protein Trait
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TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide Prokaryotic cell
LE DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide Prokaryotic cell
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transcription of DNA to mRNA starts at the promoter, ends at the terminator
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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
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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
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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)
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b - galactosidase β-galactosidase is our enzyme of choice.
This enzymatic protein is a tetramer. That means that it is comprised of how many identical subunits? The active sites of this enzyme are located at the junctions of the subunits…here…here…here…and here. This enzyme is produced by many different organisms and has been very thoroughly studied. You will be studying its production in a bacteria and its activity using enzyme purified from a mold. Transition: So what is exactly is beta-galactosidase and what does it do?… 10
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b - galactosidase galactose lactose b - galactosidase glucose H O
2 galactose Well, Humans also produce β-galactosidase though the human version of this enzyme is called lactase. Does anyone know what lactase deficiency is called? [No? ok./You’re right] Let me tell you how this enzyme works: β-galactosidase catalyzes the breakdown of a disaccharide, lactose, into two monosaccharides: glucose and galactose. Glucose is the prime energy source for many organisms. [Any ideas now?] In lactose intolerance, undigested lactose causes intestinal discomfort. Transition: We know that both the amount produced and the efficiency of individual β-galactosidase are tightly regulated, which begs the question… lactose b - galactosidase glucose (aka lactase in humans) 11
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b - galactosidase Regulation • Why Regulate b - galactosidase ? •
Levels at which b - galactosidase can be regulated: • Genetic • Biochemical Why do you think this enzyme is regulated? {-limited energy –production takes energy –environments vary –don’t waste energy}. I mentioned that both the amount and efficiency of the enzyme are regulated. Which sort of regulation do you think will happen at the genetic level? Which sort do you think will happen at the biochemical level, i.e., after the enzyme is produced? Transition: In this lab, your task will be to identify an unknown substance by examining how it regulates β-galactosidase at both the genetic and biochemical levels Caveat- as Albert pointed out, this is not regulated the same way in people as it is in E.coli. 12
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LE 18-20 Regulation of enzyme activity Regulation of enzyme production The manufacture of enzymes responsible for the biosynthesis of the amino acid tryptophan is also closely regulated Precursor Feedback inhibition Enzyme 1 Gene 1 Enzyme 2 Gene 2 Regulation of gene expression Enzyme 3 Gene 3 Enzyme 4 Gene 4 Enzyme 5 Gene 5 Tryptophan
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Polypeptides that make up enzymes for tryptophan synthesis
Prokaryotic Operon structure ensures efficient regulation of transcription trp operon Promoter Promoter Genes of operon DNA trpR trpE trpD trpC trpB trpA Operator Regulatory gene RNA polymerase Start codon Stop codon 3¢ mRNA 5¢ mRNA 5¢ E D C B A Protein Inactive repressor 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
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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
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LE 18-21b_1 DNA mRNA Protein Active repressor Tryptophan (corepressor) Tryptophan present, repressor active, operon off
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LE 18-21b_2 DNA No RNA made mRNA Protein Active repressor Tryptophan (corepressor) Tryptophan present, repressor active, operon off
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Basic Operon Regulation
Repressor Protein RNA Polymerase NO TRANSCRIPTION Promoter Operator Structural Genes Repressor mRNA Regulator Gene
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Tryptophan Operon Tryptophan Present NO TRANSCRIPTION trpR mRNA
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)
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TrpR protein (homodimer) (cannot 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)
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Tryptophan Repressor Protein
TrpR protein subunits Tryptophan (co-repressor) DNA
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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
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LE 18-22a Regulatory gene Promoter Operator DNA lacl lacZ No RNA made
3¢ mRNA RNA polymerase 5¢ Active repressor Protein Lactose absent, repressor active, operon off
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LE 18-22b lac operon DNA lacl lacZ lacY lacA RNA polymerase 3¢ mRNA
5¢ Permease Transacetylase Protein -Galactosidase Inactive repressor Allolactose (inducer) Lactose present, repressor inactive, operon on
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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
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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
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Lactose present, glucose scarce (cAMP level high): abundant lac
LE 18-23a Promoter DNA lacl lacZ RNA polymerase can bind and transcribe CAP-binding site Operator Active CAP cAMP Inactive lac repressor Inactive CAP Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized
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Lactose present, glucose present (cAMP level low): little lac
LE 18-23b Promoter DNA lacl lacZ 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
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What about the lux operon?
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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.
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Operons can be cut and pasted together to make operon fusions
Tryptophan Operon Lactose Operon Repressor Pro. Oper. Att. TrpE, D, C, B, A lacI T Pro. Oper. Z gene Y gene A gene Promoter Operator Z gene Y gene A gene mRNA β-gal mutant trpR-containing plasmid
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If the repressor is knocked out, what will happen in the presence of Tryptophan?
Tryptophan Operon Lactose Operon Repressor Pro. Oper. Att. TrpE, D, C, B, A lacI T Pro. Oper. Z gene Y gene A gene Promoter Operator Z gene Y gene A gene mRNA β-gal
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What if we add a plasmid which contains the TrpR gene. With tryptophan
What if we add a plasmid which contains the TrpR gene? With tryptophan? Without? Tryptophan Operon Lactose Operon Repressor Pro. Oper. Att. TrpE, D, C, B, A lacI T Pro. Oper. Z gene Y gene A gene Promoter Operator Z gene Y gene A gene mRNA β-gal mutant trpR-containing plasmid
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Another engineered plasmid with fusion Operon: pGLO
HELWIG 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… pGLO ori bla GFP araC 4/12/2017 VandePol
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Is this: Anabolic or Catabolic. Positive or negative
Is this: Anabolic or Catabolic? Positive or negative? Inducible or repressible? 4/12/2017
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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”?
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Which colonies will glow?
Grow? Glow? Which colonies will glow? Follow protocol On which plates will colonies grow? Which colonies will glow? LB/Amp LB/Amp/Ara LB 4/12/2017
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Appendix: pGLO slides that may be helpful
Stuff about GFP, arabinose, beta-lactamase, etc.
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DNA polymerase binds to the ori
LE 16-12 Parental (template) strand 0.25 µm Origin of replication Daughter (new) strand Bubble 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).
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On pGLO, the regulatory regions of the Arabinose operon have been glued to the structural sequences for GFP Gene Regulation RNA Polymerase araC ara GFP Operon GFP Gene Effector (Arabinose) B A D araC RNA Polymerase Effector (Arabinose) ara Operon What will happen on the Ara (+) plates? What will happen on the Ara (-) plates?
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Using GFP as a biological tracer
GFP can be fused to cellular proteins Using GFP as a biological tracer With permission from Marc Zimmer
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The pGLO plasmid ori- origin of replication
GFP- green fluorescent protein bla- Beta-lactamase araC- Arabinose What are all the other marks?
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Beta- lactam antibiotics have a similar structure
Includes penicillin, ampicillin, and others The beta-lactam ring is a square structure common to all
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Beta-lactamase can destroy a beta-lactam ring
Breaking the ring destroys the antibiotic’s effectiveness
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What about araC? Arabinose is a 5-carbon sugar, different from ribose
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