1. Chapter 7 Operons: Fine Control of Bacterial Transcription
Student learning outcomes:
Explain basic features of regulation of operons:
classic catabolic lac and ara; anabolic system trp.
role of repressors, operators, positive controls
Describe briefly role or riboswitches in regulation
Lac repressor (pink) and CAP-cAMP (blue) binding lac operator/promoter region

2. Bacterial transcription
Regulation of gene expression at transcriptional level is very important for prokaryotes:
- conserves energy, permits rapid response
- mRNA in prokaryotes is short-lived
- regulation involves protein-ligand and protein-DNA
interactions primarily
Important Figs: 1, 2, 3*, 5*, 6, 7, 10, 11, 12, 13, 14, 15*, 16, 18, 19, 20, 21*, 26, 27*, 28, 29, 31, 32, 34
Review Q: 2, 3, 4, 5, 10, 11, 13, 16, 19, 21
Analyt Q: 1, 2, 3, 4

3. 7.1 lac Operon of E. coli: catabolism of lactose
First operon discovered (Jacob & Monod 1940s-1950s)
3 genes coding for proteins to use of sugar lactose
Galactoside permease (lacY) transports lactose into cells
b-galactosidase (lacZ) cleaves lactose into galactose and glucose
Galactoside transacetylase (lacA) function unclear
Fig. 1

4. Genes of the lac Operon Genes adjacent on chromosome
lacZ = b-galactosidase
lacY = galactoside permease
lacA = galactoside transacetylase
3 genes transcribed as 1 mRNA, polycistronic message that starts from one promoter
Each cistron, or gene, has own ribosome binding site
Each cistron can be translated by separate ribosomes

5. Control of lac Operon
Glucose preferred substrate for E. coli; only make lac enzymes if need to use lactose as carbon source
lac operon is tightly controlled, 2 types of control:
Negative control for catabolic systems:
‘transcription is off unless turn on’;
inducer must remove repressor from operator
Activator, positive factor CAP protein:
responds to low glucose (energy) by stimulating transcription of lac operon

6. Negative Control of the lac Operon
‘Off until needed to turn on’
Off-regulation by lac repressor:
Product of lacI gene
Tetramer of 4 identical polypeptides
Binds operator just upstream of promoter
When repressor binds operator, operon is repressed
Repressor bound to operator prevents RNA polymerase from initiating at promoter
lac operon is repressed as long as no lactose is available or needed (i.e. glucose available)

7. Negative Control of the lac Operon
Fig. 7.3

8. Inducer of the lac Operon
Repressor is an allosteric protein
Binding of molecule (inducer) to one place on protein changes shape of a different site on the protein
Alters its interaction with a second molecule (DNA)
Inducer binds each monomer of repressor
Repressor changes conformation to favor release from operator (the other molecule)
Allolactose, the inducer, is alternative form of lactose
Fig. 7.4

9. Discovery of the lac Operon
1940s -1950s, Jacob & Monod studied metabolism of lactose by E. coli: biochemistry, genetics: cis/trans tests, antibodies to b-galactosidase, synthetic non-cleavable inducers
Three enzyme activities / three genes were induced together by galactosides
Constitutive mutants need no induction; genes are active all the time
Merodiploids, partial diploid bacteria, constructed by conjugation of F’lac into F- lac cells, were critical to analysis of mutants, to decipher cis/trans relationships to define proteins, DNA sites

10. Effects of Regulatory Mutations: Wild-type and Mutated Repressors
Fig Lac I- mutations are recessive; repressor can act in trans to affect genes on both pieces of DNA

11. Effects of Regulatory Mutations: Wild-type and Mutated Operators (with Defective Binding)
Fig Lac Oc (constitutive) mutation is cis-dominant; affects only gene adjacent to mutant operator

12. Repressor-Operator Interactions
lac repressor binds lac operator (Filter-binding assay)
[g32P]-labeled phage lambda DNA with lac operator was mixed with protein & filtered (protein binds, plus any bound DNA)
Inducer IPTG removes repressor
Mutated constitutive lac operator has lower affinity for lac repressor

13. Regulatory Mutations: Mutated Repressors (is) Bind Irreversibly; i-d repressors are dominant to WT
Fig. 7.5 mutant repressors act in trans

14. Mechanism of lac Repression still unclear
Initial binding in vitro: repressor does not block access by RNAP to lac promoter
RNAP and repressor bind together to lac promoter
Polymerase-promoter complex is in equilibrium with free polymerase and promoter
Two hypotheses remain:
RNAP can bind lac promoter in presence of repressor
Repressor inhibits transition from abortive transcription to processive transcription
Repressor, by binding to operator, blocks access by RNAP to adjacent promoter

15. There are 3 lac Operators
Major lac operator (O1) adjacent to promoter
Auxiliary lac operators - upstream and downstream
All operators required for optimum repression (Fig. 11)
O1 alone produces only modest repression
Repressor tetramer binds 2 operators to loop DNA
Fig. 10

16. lac repressor tetramer has 2 DNA binding faces
Fig. 12 Ponzy Lu crystal structure with repressor, 21-bp O1 DNA

17. Catabolite Repression of lac Operon; cAMP
When glucose is present, lac operon relatively inactive
Selection of glucose metabolism attributed to role of some breakdown product, catabolite
Catabolite repression uses breakdown product of glucose to repress other catabolic operons
Lack of glucose increases cAMP and relieves repression
Fig. 13 5’-3’cAMP

18. Catabolite Activator Protein - CAP
cAMP added to E. coli cultures overcomes catabolite repression of lac operon:
activates lac genes even in presence of glucose
Positive controller of lac operon has 2 parts:
cAMP
Protein factor known as:
Catabolite activator protein or CAP
Cyclic-AMP receptor protein or CRP
Gene encoding this protein is crp

19. CAP-cAMP Stimulates b-galactosidase synthesis
CAP-cAMP complex positively controls amount (activity) of b-galactosidase
CAP binds cAMP tightly
Mutant CAP does not bind cAMP tightly
Compare activity and production of b-galactosidase
Low activity with mutant CAP-cAMP
Fig. 14 cell extracts of cultures

20. Mechanism of CAP Action
CAP-cAMP complex binds lac promoter tightly
Mutants whose lac gene not stimulated by cAMP had mutations just upstream of lac promoter
Ex. L1 deletion mutant
Binding of CAP and cAMP to activator site helps RNAP form open promoter complex (RPo)
CAP-cAMP activated operons have weak promoters
The -35 boxes are unlike consensus sequence
If promoters were strong, could be activated even when glucose is present
Fig. 7.16

21. CAP Plus cAMP Action
RPo does not form even if RNAP has bound DNA, unless CAP-cAMP complex is also bound
Rifampicin used to prevent reinitiation
Fig. 15

22. CAP-cAMP Recruits RNAP
Two steps:
Formation of closed promoter complex RPo
Conversion of RPo into open promoter complex
CAP-cAMP bends target DNA about 100° when binds
DNA red; CAP blue; cAMP thin pink; a green
CAP contacts a subunit of RNAP (Fig. 17a)

23. CAP-cAMP Complexes bend promoter DNA
Measure protein binding to [g32P]-DNA using native PAG:
Bent DNA runs slower than linear;
Use fragments with CAP binding site (red) in different positions relative to restriction enzyme sites
See which fragment runs slowest
Fig. 18; experimental agreed with predictions

24. Model of CAP-cAMP Activation of lac Transcription
CAP-cAMP dimer binds to target site on DNA
The aCTD (carboxy terminal domain of a subunit) of RNAP interacts with specific site on CAP
Protein-protein interactions important, as are protein-DNA
Strengthens binding between promoter and RNAP

25. 7.2 The ara Operon of E. coli (ara CBAD)
ara operon encodes enzymes (genes B,A,D) required to metabolize sugar arabinose
Catabolite-repressible operon
Two ara operators:
araO1 regulates transcription of control gene araC
araO2 far upstream of promoter PBAD it controls
CAP-binding site 200 bp upstream of PBAD promoter; yet CAP stimulates transcription
Negative regulation mediated by AraC control protein

26. ara Operon Repression Loop
The araO2 operator controls transcription from PBAD promoter 250 downstream
Data suggests DNA between operator and promoter loop out; proteins bind same face of DNA
Mutant constructs changing spacing – 1 turn or ½ turn helix
Fig. 20

27. AraC, the ara Control Protein
AraC, is both positive and negative regulator
AraC has 3 binding sites:
Far upstream site (-250), araO2
araO1 located between -106 and -144
araI is 2 half-sites, each bind one araC monomer:
araI1 between -56 and -78
araI to -51
Fig. 7.21

28. Control of the ara Operon
Fig. 21
- glucose

29. AraC loops DNA by binding two operators
Add AraC to labeled minicircles with wt or mutant AraC binding sites -> looping makes more supercoiled and moves faster;
At to, add excess unlabeled DNA of strong araI site;
Electrophorese to see if still looped; mutants bind weaker
Fig. 22

30. Positive Control of the ara Operon
Positive control is also mediated by CAP and cAMP
CAP-cAMP complex attaches to binding site Pc upstream of araBAD promoter
DNA looping would allow CAP to contact a subunit of RNAP, stimulate its binding to the promoter
Fig. 7.21
- glucose

31. ara Operon Summary ara operon controlled by AraC protein
Represses by looping out DNA between araO2 and araI1 that are 210 bp apart
Arabinose derepresses operon by binding AraC; AraC loosens attachment to araO2 and binds araI2
Breaking loop allows transcription of operon from PBAD
CAP-cAMP stimulate transcription(bind upstream araI)
AraC controls own synthesis by binding araO1 to prevent leftward transcription of araC gene from Pc
Fig. 7.26

32. 7.3 The trp Operon – anabolic - tryptophan
E. coli trp operon contains 5 genes (E,D,C,B,A) for enzymes to make amino acid tryptophan (anabolic)
Anabolic enzyme synthesis typically on, and turned off by high level of product of pathway (repressed)
Operon subject to negative control of transcription by repressor when high tryptophan levels
The trp operator lies wholly within trp promoter
Tryptophan helps trp repressor bind operator
trp operon also exhibits attenuation of transcription

33. Negative Control of trp Operon
Without tryptophan no trp repressor exists, just inactive aporepressor
If aporepressor binds tryptophan, changes conformation to gain high affinity for trp operator
Aporepressor plus tryptophan makes trp repressor
Tryptophan is corepressor
Fig. 27

34. Attenuation of transcription in trp Operon extra level of control than just repressor-operator
Fig. 28: Attenuation is premature termination in 5’ UTR of trp E

35. Mechanism of Attenuation – trptophan high
Premature termination of operon’s transcript when product trp is abundant;
Involves mRNA secondary structures:
Hairpin plus string of U in leader signals termination, release of short RNA
Fig. 29

36. Defeating Attenuation – tryptophan low
Prokaryotes start making protein before mRNA completely finished – only one cell compartment.
If amino acid supply is low, ribosomes stall at tandem tryptophan codons in trp leader peptide
stalled protein synthesis ->
ribosome position influences mRNA folding
Prevents formation of hairpin (part of transcription termination signal which causes attenuation)
Fig Trp leader has tandem trp codons

37. Overriding Attenuation – low tryptophan
Ribosomes stall at tandem tryptophan codons in leader peptide
Stalled ribosome position influences way mRNA folds
Prevents formation of hairpin; hairpin part of transcription termination signal which caused attenuation if lots of trp and translation of leader.
Fig. 7.32

38. 7.4 Riboswitches
Small molecules can act directly on 5’-UTRs of mRNAs to control gene expression
Riboswitches –regions of 5’-UTRs that can alter structure to control gene expression after bind ligand
Region that binds ligand is called aptamer
Expression platform is other module in riboswitch:
Terminator
Ribosome-binding site
Another RNA element that affects gene expression
Operates by depressing gene expression
Some work at transcriptional level
Others can function at translational level

39. Model of Riboswitch Action: ribD gene for riboflavin synthesis
FMN binds to aptamer in called RFN element in 5’-UTR of ribD mRNA
Binding FMN, base pairing in riboswitch changes to create terminator
Transcription is attenuated
Saves cell energy as FMN is product of the ribD operon
Fig. 34 Model for B. subtilis ribD gene for riboflavin synthesis, the flavin mononucleotide FMN

40. Riboswitch Action: ribD gene
Excess FMN changes shape of 5’ mRNA leader
Structure probed with RNase T1, OH- cleavage; -/+ FMN
Fig. 7.33

41. Review problems
5. Describe and give results of experiment that shows lac operator is site of repressor binding.
21. Why does translation of the trp leader region not simply continue into the trp structural genes (trpE, etc.) in E. coli ?
AQ 3. Consider E. coli cells each having one of the following mutations: Indicate effect each mutation on function of lac operon (assuming no glucose is present):
a. mutant lac operator (Oc locus) that doesn’t bind repressor.
b. mutant lac repressor (I-) that doesn’t bind operator
c. Mutant lac repressor (Is) that doesn’t bind inducer
d. Mutant lac promoter that doesn’t bind CAP-CAMP

42. Review problems
4. Why are negative and positive control of the lac operon important to the neergy efficiency of E. coli cells?
14. Diagram how arabinose relieves repression in the araBAD operon. Show whereAraC is located (a) in the absence of arabinose; (b) in the presence of arabinose and lack glucose.
See Fig. 31. Because the his operon for synthesis of histidine operates much like trp operon. What might you predict about the sequence of amino acids encoded by the his leader?