1. MICB404, Spring 2008 Lecture #25 Operons
Microbial Genetics
MICB404, Spring 2008
Lecture #25
Operons

2. Announcements Today’s lecture
Spring break!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
Today’s lecture
lac operon

3. Operons
“functionally integrated genetic units for control of gene expression. Consist of structural genes, and of adjacent sites (promoter and operator) that control transcription of the structural genes”
structural genes involved in shared metabolic pathways, protein complexes, or cellular functions
coordinated regulation of gene expression
transcriptional initiation (repression, activation)
transcriptional termination
post-transcriptional regulation
mRNA stability, translation
protein stability, activity

4. lac operon
(lactose, allolactose, etc.)
LacI

5. Complementation and cis/trans tests
Dominant vs recessive mutations
- recessive: does not exert phenotype if wildtype allele is present
- dominant: exerts phenotype even if wildtype allele is present
cis vs. trans acting mutations
- cis: typically on a non-coding regulatory DNA (affects only that DNA and can’t be complimented)
- trans: usually affects a diffusible gene product (can be complimented)

6. lac operon genetics Complementation and cis/trans tests
F’ plasmid bearing lac mutations
Conjugate with recipient bearing chromosomal lac mutations
Score phenotype of transconjugant

7. lac operon genetics lacI- lacZ+ lacY+ lac A+ Operon 1 Operon 2
Inducer present
Inducer absent
Recessive or dominant?
cis or trans?

8. lac operon genetics lacI+ lacOc lacZ+ lacY+ lac A+ Operon 1 Operon 2
lacI+ lacO+ lacZ+ lacY+ lac A+
Repressor binds only partially to Oc
Inducer present
Inducer absent

9. lac operon genetics lacI+ lacOc lacZ+ lacY+ lac A+ Operon 1 Operon 2
lacI+ lacO+ lacZ- lacY+ lac A+
Repressor binds only partially to Oc
Inducer present
Inducer absent

10. lac operon genetics lacI+ lacOc lacZ- lacY+ lac A+ Operon 1 Operon 2
lacI+ lacO+ lacZ+ lacY+ lac A+
Repressor binds only partially to Oc
Inducer present
Inducer absent

11. lac operon genetics lacI+ lacOc lacZ- lacY+ lac A+ Operon 1 Operon 2
lacI+ lacO+ lacZ+ lacY+ lac A+
As long as there is a functional lacZ gene adjacent to a Oc operator, β-galactosidase expression will be observed even if a wildtype operator is present in the same cell.
cis or trans?
Recessive or dominant?

12. Summary of lac mutations
Expression
Relation to wildtype
Action I+
Inducible
trans I-
Constitutive
Recessive IS
Non-
Dominant ID OC
(partially)
cis

13. lac operon genetics
Mutation in lacZ and lacY more common than in lacO or lacP
target size
lacO is divided into 3 operator sites
LacI as tetramer binds to them can bind to them indi- vidually or in pairs
bent promoter unable to recruit RNA polymerase

14. Catabolite repression
Glucose is carbon source of choice for E. coli
Diauxic growth: glucose is completely consumed before lactose consumption begins

15. Diauxic growth
Diauxic shift
[lactose]
[glucose]

16. Catabolite repression
high [glucose]  low [cAMP]i
As glucose is consumed, intracellular [cAMP] increases
ATP cAMP
adenylate cyclase (cya)

17. Catabolite repression
high [glucose]  low [cAMP]I
how?
Glucose uptake via PhosphoTransferase System (PTS)
One component of PTS (IIAGlc) is dephosphorylated upon glucose uptake
IIAGlc-PO4 activates adenylate cyclase
If IIAGlc is participating in glucose transport, it is not able the activate adenylate cyclase

18. Catabolite repression
cAMP binds with CAP protein
“catabolite activator protein”
also called CRP
CAP-cAMP binds to lac promoter
increases efficiency of RNA polymerase transcriptional initiation

19. Catabolite repression
High glucose  low cAMP  low transcription
High lactose  LacI released from operator
Low glucose + high lactose  expression of lac operon
Sugars in growth medium
β-galactosidase activity (relative)
glucose 1
glucose + lactose 50
lactose
2500

20. Catabolite repression
Also acts on mal, gal, ara operons
and others
Global Regulatory Mechanism

21. Inducer exclusion Glucose also prevents uptake of lactose from medium
Component of PTS binds to LacY and inhibits lactose uptake
By excluding the inducer from the cell, expression of the operon is further repressed

22. Catabolite repression
[cAMP] low
CAP-cAMP doesn’t bind to lacP and transcription not activated

23. Catabolite repression
[cAMP] high
CAP-cAMP binds to lacP and transcription potentially activated
If lactose is present, repression is removed and lac operon expressed

24. CAP protein Contacts RNA polymerase
Together, these result in activation of lac operon transcription

25. trp operon
Tryptophan is required for protein synthesis and hence growth
E. coli can use exogenous Trp
When unavailable, synthesis is required
trp operon
Five structural genes encoding three enzymes required to convert chorismic acid into tryptophan

26. trp operon
Anthranilate synthetase – trpE, trpD
N-(5’-phosphoribosyl)-anthrnilate isomerase/Indole-3-glycerol phosphate synthase – trpC
Trytophan synthase – trpB, trpA
When trp repressor bound to operator, transcription does not occur

27. trp operon regulation
Expression of trpEDCBA is reduced by the addition of tryptophan in trpR mutants
Lead to discovery of a 2nd level of trytophan control
- attenuation

28. trp operon regulation Attenuation - tryptophanyl-tRNATrp
tRNATrp charged with tryptophan
- trpL gene, a non-coding leader sequence at the 5’ end

29. trp operon regulation
string of T’s (in DNA)
stem-loop

30. trp operon regulation Base-pairing
Stem-loops 1-2 and 3-4 (terminator), or
Stem-loop 2-3 (anti-terminator)

31. trp operon regulation
Translation of leader peptide deter-mines which stem-loops form
Transcription and translation are coupled
RNA:RNA base pairing is eliminated in region in contact with ribosome
Formation of stem-loops 1-2 and 3-4 is mutually exclusive of formation of stem-loop 2-3

32. trp operon regulation Trp codons make translation sensitive to [Trp]
At low [Trp], translation stalls at Trp codons
Stem-loop 2-3 forms
Transcription does not terminate
trp enzymes expressed

33. trp operon regulation
At high [Trp], leader peptide translated completely
Ribosome contacts segment 2, preventing formation of stem-loop 2-3
Stem-loop 3-4, transcriptional terminator, forms
trp enzyme mRNA is not transcribed

34. trp operon regulation Fine control
At intermediate [Trp] abundance only some ribosomes will stall resulting in low-level expression of Trp biosynthetic enzymes
Interplay between TrpR repressor activity and attenuation
Attenuation mechanisms common for amino acid biosynthetic operons
His, Thr, Leu, Phe, Ile-Val

35. ara operon Regulated by araC gene product
araO1, AraC binds and represses its own transcription. With arabinose, AraC bound at this site activates PBAD.
araO2, AraC bound at this site can simultaneously bind to the araI site to repress PBAD.
araI, is also an inducer site. In the presence of arabinose, AraC bound at this site helps activate PBAD.
CAP binding, when arabinose is present, it promotes the rearrangement of AraC (repression of PBAD to activation of PBAD)

36. ara operon
Arabinose: 5-C sugar
cAMP
CAP
Pentose Phosphate
Pathway

37. ara operon AraC binds arabinose P1 state: no arabinose bound
P2 state: arabinose bound, and functions as transcriptional activator

38. ara operon
In absence of arabinose, AraC (P1 state) binds to araI1 and araO2
DNA loops out and RNA polymerase cannot access pBAD

39. ara operon
arabinose
In presence of arabinose, AraC (P2 state) is induced to bind araI and 01
Operon’s pBAD promoter becomes accessible
CAP-cAMP helps AraC assemble on activator sites and activate transcription from pBAD

40. ara operon AraC autoregulation
at low arabinose, AraC binds araO2 and araI1
and prevents araC transcription
at high [AraC], it binds to araO1

41. ara operon Regulation Autoregulation of araC
cAMP/CAP and catabolite repression
Activation of AraC by conformational change upon arabinose binding
Alteration of AraC activity by adjacent binding of cAMP-CAP

42. Spring break
Study hard!