1. Control of Gene Expression:
Prokaryotes
BIOL 105
Dr. Corl
October 30, 2013

2. Gene Expression
• We can say that Gene “A” is being expressed if:
– Gene “A” is being transcribed to form mRNA
– That mRNA is being translated to form proteins
– Those proteins are properly folded and are in state
where they can be used by the cell
• Cells are extremely selective about what genes
are expressed, in what amounts, and when.
• Gene expression can be regulated at a variety of
different stages.

3. Regulation of Gene Expression
• Gene expression can be regulated:
– During transcription (transcriptional control).
– During translation (translational control).
– After translation (post-translational control).

4. Some Examples of
Regulation of Gene Expression
• Transcriptional control:
– Regulatory proteins affect the ability for RNA
polymerase to bind to or transcribe a particular gene.
• Translational control:
– Various proteins may affect rate of translation.
– Enzymes may affect lifetime of an mRNA transcript.
• Post-translational control:
– Translated protein may be modified by phosphorylation,
which may change its folding and/or activity.

5. Gene Expression
• Some genes are constitutively expressed:
– Expressed equally at all times.
• Many other genes are regulated and their
expression may be induced or repressed:
– Gene expression is not just “on” or “off” it can vary along a continuum!
• Today we will be looking at transcriptional
regulation of gene expression in bacteria.

6. Lactose Metabolism in E. coli
• Bacteria break down lactose into its component
monomers, glucose and galactose, using the
enzyme β-galactosidase.
• Bacteria can then use the monomers to
generate ATP through cellular respiration.

7. Lactose Metabolism in E. coli
• Lactose gets transported (imported) into the cell
with the help of galactoside permease.
• Once inside the cell, lactose can be cleaved into
monomers with the help of β-galactosidase.

8. Lactose Metabolism in E. coli
• The expression of the gene for β-galactosidase
(and galactoside permease) is regulated by
lactose and glucose:
– High levels of LACTOSE stimulate (induce) the
expression of the gene for β-galactosidase.
– High levels of glucose inhibit (repress) the
expression of the gene for β-galactosidase.
• How (by what mechanisms) do lactose and
glucose regulate gene expression in E. coli?

9. Lactose Metabolism Mutants
• Monod and Jacob conducted a
genetic screen for E. coli mutants
that were specifically defective in
lactose metabolism:
– Generated a large number of E. coli
colonies bearing mutations in random
locations in their genomes.
– “screened” through these colonies to find
ones that could not successfully grow on
a “lactose only” medium, but could grow
on a “glucose only” medium.

10. Screen Results
• Three classes of E. coli mutants defective in
lactose metabolism were found:
– 1.) lacZ- mutants lacked functional β-galactosidase.
– 2.) lacY- mutants lacked the membrane protein
galactosidase permease.

11. Screen Results
• 3.) lac I- mutants:
– Produce β-galactosidase and galactoside permease even
when lactose is absent.
– lacI- mutants are constitutive mutants:
• They produce product(s) at all times.
• Loss of regulation by lactose.
– The lacI gene must code for a protein that normally
serves to repress expression of the lacZ and lacY genes
when lactose is absent.

12. Screen
Conclusions
• The lacZ gene codes for:
β-galactosidase.
• The lacY gene codes for:
– Galactoside permease.
• The lacI gene codes for:
– A regulatory protein (a repressor) that normally
functions to repress lacZ and lacY gene
expression when lactose is absent.

13. The lac Genes
• The lacZ, lacY, and lacI genes are in close
physical proximity to each other on the
bacterial chromosome.
• How does the lacI gene product repress gene
expression of lacZ and lacY?

14. Negative Control of Transcription
• Negative control occurs when:
– A regulatory protein (a repressor) binds to DNA and
decreases the rate of transcription of downstream genes.
• The lacI gene codes for a repressor, a regulatory protein
that exerts negative control over the lacZ and lacY genes.

15. Positive Control of Transcription
• Positive control occurs when:
– A regulatory protein (an _______) binds to DNA and
________ the rate of transcription of downstream genes.
• We haven’t encountered this yet, but we will by the end of
this lecture. Stay tuned!

16. Negative Control of
lacZ and lacY Gene Expression
• The lacI gene codes for a repressor that
binds to DNA just downstream of the
promoter, physically blocking
transcription of lacZ and lacY.

17. Negative Control of
lacZ and lacY Gene Expression
• In the presence of lactose:
–
–
–
–
Lactose binds to the repressor.
Lactose-repressor complex releases from DNA.
RNA polymerase can now transcribe lacZ and lacY.
Thus, lactose induces transcription by preventing the
repressor from exerting negative control.

18. Negative Control of
lacZ and lacY Gene Expression
• If the lacI gene is mutated (lacI- mutants):
– No functional repressor is synthesized.
– No repressor is ever present in the cell to bind
downstream of the promoter to block transcription.
– lacZ and lacY will be transcribed regardless of
whether lactose is present or absent.

19. Summary Thus Far

20. The lac Operon
• Operon:
– A set of coordinately regulated bacterial genes.
• The lac operon:
– A group of genes involved in lactose metabolism.
– Also includes the lacA gene, which codes for a
protective enzyme, transacetylase.
– Encodes a polycistronic mRNA:
• mRNA that contains >1 protein encoding segment.
– The lacI protein is a repressor that binds to the
operator region of the lac operon.
– Lactose is an inducer, binding to the lacI repressor
protein and causing it to release from the operator.

21. The trp Operon
• Contains 5 genes coding for proteins (enzymes)
required for the synthesis of the amino acid
tryptophan. Also contains a promoter and operator.
• When tryptophan levels in the cell are low, the
expression of the trp operon genes is relatively high.
– There are trp repressor proteins in the cell, but they are
unable to bind to the trp operon operator all on their own.

22. The trp Operon
• When tryptophan levels in the cell are high:
– Tryptophan binds to the repressor and activities it.
– The activated trp repressor can now bind to the trp
operator and block transcription of the trp operon genes.

23. The lac
and trp
Operons

24. Positive Control of Transcription
• Occurs when a regulatory protein (an activator) binds to DNA
and increases the rate of transcription of downstream genes.
• Let’s look at two examples of activator proteins at work:
– AraC proteins can increase transcription of the ara operon genes.
– CAP proteins can increase transcription of the lac operon genes.

25. Positive Control of the ara Operon
• E. coli can also utilize arabinose, a pentose
found in plant cell walls, as an energy source.
• The ara operon includes:
– 3 genes required for arabinose metabolism.
– A promoter to which RNA polymerase can bind.
– An initiator sequence to which an activator protein
(AraC) can bind to stimulate transcription.

26. Positive Control of the ara Operon
• Expression of the ara operon genes is high only
when arabinose levels are high:
– (If you were E. coli, you wouldn’t want to waste
precious resources expressing these genes unless
there was arabinose around to break down!)
– How does the presence of arabinose stimulate
expression of the ara operon genes?

27. Positive Control of the ara Operon
• If arabinose levels are relatively high…
– Arabinose binds to AraC proteins in the cell…
– ….which allows the AraC proteins to bind to the initiator
region of the ara operon….
– ….which helps RNA polymerase to bind to the promoter
region of the ara operon successfully…
– …which increases transcription rate of ara operon genes.

28. Positive Control of the ara Operon
• Thus, AraC proteins, when bound to arabinose, can act as
activators of gene expression at the ara operon:
– Example of positive control of gene expression.
• Let’s look at one more example of positive control of gene
expression, this time going back to the lac operon.
– Gene expression at the lac operon is under both:
• Negative control (by the lacI repressor protein)
• Positive control (by a protein called CAP)

29. Positive Control of the lac Operon
• Binding of CAP (an activator protein) to the
CAP site (a DNAregion) just upstream of the
lac operon promoter greatly increases lac
operon gene expression.
• CAP can only bind the CAP site if CAP is
bound to cAMP (cyclic AMP).

30. Positive Control of the lac Operon
• If cAMP levels are low, CAP is inactive
and won’t bind to the CAP site, and
therefore transcription will be infrequent.
• What regulates cAMP levels?

31. Glucose Regulates cAMP Levels
• cAMP is synthesized from ATP by an enzyme
called adenylyl cyclase.
• Glucose inhibits adenylyl cyclase activity.
• If glucose levels are high, this will cause
cAMP levels in the cell to be low.

32. Glucose Regulates cAMP Levels
• In this way, glucose, by regulating
cAMP levels, can regulate the
expression of the lac operon genes.

33. Another Effect of
Glucose
• Recent studies have revealed a second, perhaps
even more significant, contribution of glucose to
regulating lac operon expression:
– High level of glucose leads to a dephosphorylation and
inactivation of gal. permease in E. coli cell membrane…
– …which inhibits lactose transport into the cell…
– ….which prevents lactose accumulation inside the cell…
– …which allows the lacI repressor protein to bind to the lac
operator, decreasing lac operon gene expression.

34. Summary: The lac Operon

35. Review Questions
• What is meant by “gene expression?” At what
levels can gene expression be controlled?
• Draw out the lac operon. How is it negatively
controlled? Positively controlled?
• Contrast the regulation of the trp operon versus
the regulation of the lac operon.
• How is the ara operon regulated by arabinose
and the AraC protein?