bio 101a

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 symbiosisSpencer V. Nyholm & Margaret McFall-NgaiNature Reviews Microbiology 2, 632-642 (August 2004)

Enzyme Regulation maintains Efficiency

9

• Enzymes: General Background

• -galactosidase

• Gene Regulation

• Biochemical Regulation

• Experiment

-galactosidase

10

-galactosidase

11

lactose

galactose

glucose

H2O

- galactosidase(aka lactase in humans)

-galactosidase Regulation

12

• Why Regulate - galactosidase ?

• Levels at which - galactosidase can be

regulated:

• Genetic

• Biochemical

The lac operon regulates when β-galactosidase is made

When lactose is present, transcription is activated

LE 18-20

Regulation of enzymeactivity

Regulation of enzymeproduction

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

Feedbackinhibition

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

Promoter Promoter

DNA trpR

Regulatorygene

RNApolymerase

mRNA

3

5

Protein Inactiverepressor

Tryptophan absent, repressor inactive, operon on

mRNA 5

trpE trpD trpC trpB trpA

OperatorStart codon Stop codon

trp operon

Genes of operon

E

Polypeptides that make upenzymes 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

LE 18-21b_1

DNA

Protein

Tryptophan(corepressor)

Tryptophan present, repressor active, operon off

mRNA

Activerepressor

LE 18-21b_2

DNA

Protein

Tryptophan(corepressor)

Tryptophan present, repressor active, operon off

mRNA

Activerepressor

No RNA made

Basic Operon Regulation

Regulator Gene

Promoter Operator Structural Genes

RNA Polymerase

Repressor mRNA

Repressor Protein

NO TRANSCRIPTION

Tryptophan OperonTryptophan Present

Regulator Gene Promoter Operator Structural GenesAttenuator

trpR 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 Gene Promoter Operator Structural GenesAttenuator

trpR 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

Regulatorygene

mRNA

5

3

RNApolymerase

ProteinActiverepressor

NoRNAmade

lacZ

Promoter

Operator

Lactose absent, repressor active, operon off

LE 18-22b

DNA lacl

mRNA5

3

lac operon

Lactose present, repressor inactive, operon on

lacZ lacY lacA

RNApolymerase

mRNA 5

Protein

Allolactose(inducer)

Inactiverepressor

-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

ActiveCAP

InactiveCAP

RNApolymerasecan bindand transcribe

Operator

lacZ

Inactive lacrepressor

Lactose present, glucose scarce (cAMP level high): abundant lacmRNA synthesized

LE 18-23b

DNA lacl

CAP-binding site

Promoter

RNApolymerasecan’t bind

Operator

lacZ

Inactive lacrepressor

InactiveCAP

Lactose present, glucose present (cAMP level low): little lacmRNA 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

lacIRepressor Oper. TrpE, D, C, B, AAtt.

Tryptophan Operon Lactose OperonT Pro. Oper.

Promoter

Operator Z gene

Y gene A gene

mRNA

β-gal

Pro. Z gene Y gene A gene

mutant trpR-containing

plasmid

If the repressor is knocked out, what will happen in the presence of Tryptophan?

lacIRepressor Oper. TrpE, D, C, B, AAtt.

Tryptophan Operon Lactose OperonT Pro. Oper.

Promoter

Operator Z gene

Y gene A gene

mRNA

β-gal

Pro. Z gene Y gene A gene

What if we add a plasmid which contains the TrpR gene? With tryptophan? Without?

lacIRepressor Oper. TrpE, D, C, B, AAtt.

Tryptophan Operon Lactose OperonT Pro. Oper.

Promoter

Operator Z gene

Y gene A gene

mRNA

β-gal

Pro. Z gene Y gene A gene

mutant trpR-containing

plasmid

04/22/23 VandePol 35

Manufactured by a private corporation

AraC- arabinose gene

GFP- Green Fluorescent protein

bla- Beta-lactamase ori- you know this…

pGLOori

blaGFP

araC

Another engineered plasmid with fusion Operon: pGLO

04/22/23 36

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”?

04/22/23 38

RNA Polymerase

Z Y A

Z Y ALacI

Effector (Lactose)

Z Y ALacI

lac Operon

B A DaraC

B A DaraC

RNA Polymerase

Effector (Arabinose)

araC B A D

ara Operon

lac Operon and ara operon

04/22/23 39

Grow? Glow?

Follow protocol On which plates will

colonies grow? Which colonies will

glow?

Which colonies will glow?

Appendix: pGLO slides that may be helpful

• Stuff about GFP, arabinose, beta-lactamase, etc.

LE 16-12

In eukaryotes, DNA replication begins at may sitesalong the giant DNA molecule of each chromosome.

Two daughter DNA molecules

Parental (template) strand

Daughter (new) strand0.25 µm

Replication fork

Origin of replication

Bubble

In this micrograph, three replicationbubbles are visible along the DNAof 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)

B A DaraC

B A DaraC

RNA Polymerase

Effector (Arabinose)

araC B A D

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?

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

Using GFP as a biological tracer

http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.htmlWith permission from Marc Zimmer

GFP can be fused to cellular proteins

GFP Fusion & Protein Localization1

2

Gene X (no stop codon)Control Region GFP gene (in-frame with Gene X)

express gene-of-interest

GFPProtein X

3Translated Fusion Protein Fluorescence Visualization

Gene Fusion

Fusion protein in C. elegans highlights nervous system

Vesicle Transport in the Caenorhabditis elegans Nervous System

SNB-1::GFP Fusion1

2

For snb-1Control Region GFP gene (in-frame with snb-1)

express gene-of-interest

3Neurotransmitter-Packaging Vesicles Present GFP-Tagged SNB-1 on Surface

View SNB-1 (Vesicle) Distribution

Fusion of snb-1 to GFP gene

GFPSynaptobrevin

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 beta-lactam ring

Breaking the ring destroys the antibiotic’s effectiveness

What about araC?

Arabinose is a 5-carbon sugar, different from ribose