1. 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?

2. 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).

3. This metabolic control occurs on two levels:
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

4. 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

5. 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

6. Polypeptides that make up enzymes for tryptophan synthesis
LE 18-21a
trp operon
Promoter
Promoter
Genes of operon
DNA
trpR
trpE
trpD
trpC
trpB
trpA
Operator
Regulatory
gene
RNA
polymerase
Start codon
Stop codon 3¢ 5¢
mRNA 5¢
Polycistronic*
mRNA E D C B A
Protein
Inactive
repressor
Polypeptides that make up
enzymes for tryptophan synthesis
Tryptophan absent, repressor inactive, operon on
* = mRNA carries the information of several genes, which are translated into several proteins

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

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

9. Trp Operon Animation

10. 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

11. 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

12. LE 18-22b lac operon DNA lacl lacZ lacY lacA RNA polymerase 3¢ mRNA 5¢
Polycistronic
mRNA
Protein
-Galactosidase
Permease
Transacetylase
Inactive
repressor
Allolactose
(inducer)
Lactose present, repressor inactive, operon on

13. Lac Operon Animation

14. 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

15. 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

16. 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

17. 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 efficiently
Inactive
CAP
Inactive lac
repressor
Lactose present, glucose present (cAMP level low): little lac
mRNA synthesized

18. Catabolite Activator Protein Mechanism
Click on “combination of switches - the lac operon”

19. 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 conformation
acts as a repressor
binds to araI and a second operator region araO
forms a loop
this loop prevents transcription

20. Application of Operons:
Regulatory gene
Promoter for
the cluster of genes
B, A, and D
Operator
(part of the promoter)

21. Arabinose operon with in-frame foreign DNA inserted:
araC regulatory gene
Gene B
Gene A
Gene D
Gene D
Tomato gene
Tomato gene
repressor
transcription
Inducer (arabinose)
stop
start
stop
stop
start
start 3´
stop
start
translation
Polycistronic mRNA 5´
translation
translation
translation
Red Fluorescent Protein
(RFP)
Protein B
Protein A
Protein D