Download - Anticipatory Questions

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Anticipatory Questions

• 1. What might happen if an organism had its cells expressing all genes within the genome all the time?

• 2. At what levels can control of cellular activities/pathways be controlled?

• 3. Based on our discussions up to this point, what do you think the term “negative feedback” means?

• 4. What steps are involved in the initiation of prokaryotic transcription?


Learning Objectives

• understand that regulation of gene expression is a means by which to control timing and rate of generation regarding functional gene product (either RNA or polypeptide/protein).

• explain the concept of an operon in terms of components’ functions (promoter, operator, repressor, co-repressor, inducer, gene cluster, polycistronic transcript).

• compare and contrast repressible and inducible operon systems/pathways.

• compare and contrast negative versus positive regulation of operons

• apply the operon concept to gene expression as it relates to genetic engineering (specifically, our cloning and expression of the “tomato” gene).


Individual bacteria respond to environmental change by regulating their gene expression

• A bacterium can tune its metabolism to the changing environment and food sources

• This metabolic control occurs on two levels:

– Adjusting activity of metabolic enzymes

– Regulating genes that encode metabolic enzymes

LE 18-20LE 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

• In bacteria, genes are often clustered into operons, composed of

– An operator, an “on-off” switch

– A promoter

– Genes for metabolic enzymes

• An operon can be switched off by a protein called a repressor

• A corepressor is a small molecule that cooperates with a repressor to switch an operon off

LE 18-21aLE 18-21a

Promoter Promoter

DNA trpR

Regulatorygene

RNApolymerase

mRNA

3

5

Protein Inactiverepressor

Tryptophan absent, repressor inactive, operon on

Polycistronic* mRNA

trpE trpD trpC trpB trpA

Operator

Start codonStop codon

trp operon

Genes of operon

E

Polypeptides that make upenzymes for tryptophan synthesis

D C B A

5

* = mRNA carries the information of several genes, which are translated into several proteins

LE 18-21b_1LE 18-21b_1

DNA

Protein

Tryptophan(corepressor)

Tryptophan present, repressor active, operon off

mRNA

Activerepressor

LE 18-21b_2LE 18-21b_2

DNA

Protein

Tryptophan(corepressor)

Tryptophan present, repressor active, operon off

mRNA

Activerepressor

No RNA made


Trp Operon Animation

• http://bcs.whfreeman.com/thelifewire/content/chp13/1302002.html

http://bcs.whfreeman.com/thelifewire/content/chp13/1302002.html






Repressible and Inducible Operons: Two Types of Negative Gene Regulation

• A repressible operon is one that is usually on; binding of a repressor to the operator 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, which contains genes coding for enzymes in hydrolysis and metabolism of lactose

LE 18-22aLE 18-22a

DNA lacl

Regulatorygene

mRNA

5

3

RNApolymerase

ProteinActiverepressor

NoRNAmade

lacZ

Promoter

Operator

Lactose absent, repressor active, operon off

LE 18-22bLE 18-22b

DNA lacl

mRNA5

3

lac operon

Lactose present, repressor inactive, operon on

lacZ lacY lacA

RNApolymerase

Polycistronic mRNA

Protein

Allolactose(inducer)

Inactiverepressor

-Galactosidase Permease Transacetylase

5


Lac Operon Animation

http://www.sumanasinc.com/webcontent/animations/content/lacoperon.html







• 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 18-23aLE 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 18-23bLE 18-23b

DNA lacl

CAP-binding site

Promoter

RNApolymerase can’t bind efficiently

Operator

lacZ

Inactive lacrepressor

InactiveCAP

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


Catabolite Activator Protein Mechanism

• http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter18/animations.html#

• Click on “combination of switches - the lac operon”


The Arabinose Operon - A Composite of Negative & Positive Regulation

a) In the presence of arabinose:•CAP-cAMP complex and araC-arabinose complex bind to initiator region •this allows RNA polymerase to bind to the promoter •transcription begins

b) In the absence of arabinose:araC protein assumes a different conformationacts as a repressor binds to araI and a second operator region araO forms a loop this loop prevents transcription


Regulatory gene

Operator (part of the promoter)

Promoter for the cluster of genes

B, A, and D

Application of Operons:


araC regulatory gene

repressor

Inducer (arabinose)

Gene D

Arabinose operon with in-frame foreign DNA inserted:

Tomato gene

start startstart

startstop

stop

stop stop

Protein B Protein A Protein D

Red Fluorescent Protein (RFP)

Gene B Gene A Gene D Tomato gene

5´

3´

Polycistronic mRNA

transcription

translationtranslation

translationtranslation