1. Bio 101A
GE III results day

2. 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

3. 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.

4. Prokaryotic Gene Regulation Bio 101A
Operon structure and function
Positive/Negative gene regulation
Promoters, operators, repressors, inducers

5. 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

6. 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

7. 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

8. 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)

9. Enzyme Regulation maintains Efficiency•
Enzymes: General Background • b -
galactosidase •
Gene Regulation •
Biochemical Regulation •
Experiment 9

10. 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

11. b - galactosidase galactose lactose b - galactosidase glucose H O2
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

12. 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

13. The lac operon regulates when β-galactosidase is made

14. When lactose is present, transcription is activated

15. LE 18-20 Regulation of enzyme activity Regulation of enzyme production
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

16. 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

17. 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

18. LE 18-21b_1
DNA
mRNA
Protein
Active
repressor
Tryptophan
(corepressor)
Tryptophan present, repressor active, operon off

19. LE 18-21b_2
DNA
No RNA made
mRNA
Protein
Active
repressor
Tryptophan
(corepressor)
Tryptophan present, repressor active, operon off

20. Basic Operon Regulation
Repressor Protein
RNA Polymerase
NO TRANSCRIPTION
Promoter
Operator
Structural Genes
Repressor mRNA
Regulator Gene

21. 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)

22. 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)

23. Tryptophan Repressor Protein
TrpR protein subunits
Tryptophan (co-repressor)
DNA

24. 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

25. LE 18-22a Regulatory gene Promoter Operator DNA lacl lacZ No RNA made3¢
mRNA
RNA
polymerase 5¢
Active
repressor
Protein
Lactose absent, repressor active, operon off

26. LE 18-22b lac operon DNA lacl lacZ lacY lacA RNA polymerase 3¢ mRNA5¢
Permease
Transacetylase
Protein
-Galactosidase
Inactive
repressor
Allolactose
(inducer)
Lactose present, repressor inactive, operon on

27. 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

28. 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

29. 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

30. 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

31. 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.  

32. 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

33. 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

34. 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

35. 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
4/16/2017
VandePol

36. Is this: Anabolic or Catabolic. Positive or negative
Is this: Anabolic or Catabolic? Positive or negative? Inducible or repressible?
4/16/2017

37. 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”?

38. lac Operon and ara operonZ Y A
LacI
Effector
(Lactose)
lac Operon B A D
araC
RNA Polymerase
Effector (Arabinose)
ara Operon
RNA Polymerase
4/16/2017

39. 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
4/16/2017

40. Appendix: pGLO slides that may be helpful
Stuff about GFP, arabinose, beta-lactamase, etc.

41. 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).

42. 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?

43. Green Fluorescent Protein
40 Å
30 Å
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
Previous Slide: Immunofluorescence
Key Points of Current Slide:
try to know the key points on this slide well so that you’re not reading it to the class.
the absorption maximum is very close to that of the FITC fluorochrome, so in our lab we will be using the FITC filter to excite GFP
CAs: FYI
autofluorescence (when cells naturally fluoresce without the addition of a fluorochrome) occurs in the C. elegans gut; using a normal GFP filter this looks yellow, but under FITC filter looks green.
Next Slide: GFP Fusion and Protein Localization
Transition: GFP applications are slightly different from those of fluorophores that are just attached post-translationally to proteins or antibodies. Instead, the GFP gene is commonly fused to a given gene of interest.

44. Using GFP as a biological tracer
GFP can be fused to cellular proteins
Using GFP as a biological tracer
With permission from Marc Zimmer

45. GFP Fusion & Protein Localization1
Gene Fusion
Gene X (no stop codon)
Control Region
GFP gene (in-frame with Gene X)
express gene-of-interest 2
Translated Fusion Protein 3
Fluorescence Visualization
Previous Slide: GFP Background Information
Key Points of Current Slide:
1. GFP is ordinarily fused to the end of the gene encoding the protein of interest. The GFP gene has to be in-frame with the protein, which cannot have a stop codon.
2. Usually researchers create this fusion in such a way that the GFP::Protein X fusion is translated as one protein. Key is that GFP cannot alter Protein X folding, and more importantly, function. GFP will glow when excited.
3. Since GFP is part of fusion protein, it will localize wherever Protein X localizes. In this example, localizes to the inner mitochondrial matrix. Useful to test for (a) protein expression/repression and (b) distribution/localization of proteins within cells or organisms.
Next Slide: Nucleic acid stains
Transition: Finally, there are markers that are specific to nucleic acids
GFP
Protein X

46. Fusion protein in C. elegans highlights nervous system
Vesicle Transport in the Caenorhabditis elegans Nervous System
[START WEEK 1 TALK]
Again, this is a title slide that you need not spend too much time on.
You may want to excite the class by telling them that we took this picture in training, and that they will see very similar images during Week 2.
Next Slide: System Objectives review
Transition: Just to refresh your memory, we’ll begin with the system objectives again.

47. SNB-1::GFP Fusion 1 Fusion of snb-1 to GFP gene 2
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 3
View SNB-1 (Vesicle) Distribution
Previous Slide: Experimental Outline
Key Points of Current Slide:
Fuse snb-1 gene (for synaptobrevin) to GFP gene
Co-expression leads to translation of fusion protein
SNB-1::GFP will be presented on the surface of neurotransmitter-packaging vesicles.
GFP will glow wherever SNB-1 is localized in the worm (in sample picture, can see glowing nerve ring, which has a large collection of axons and dendrites)
Blue = DAPI (cell localization)
Next Slide: Transgenic worms
Transition: We used a process called transformation by injection (yeah molbio!) to introduce this fusion protein into the worms.
GFP
Synaptobrevin

48. 2008 Nobel Prize- GFP
GFP mice

49. 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?

50. Beta- lactam antibiotics have a similar structure
Includes penicillin, ampicillin, and others
The beta-lactam ring is a square structure common to all

51. Beta-lactamase can destroy a beta-lactam ring
Breaking the ring destroys the antibiotic’s effectiveness

52. What about araC?
Arabinose is a 5-carbon sugar, different from ribose