1. Operons: Fine Control of Bacterial Transcription
Chapter 7

2. Why is Operon system important?
3000 genes in E.coli – some products are in great demand
Some genes are turned off – rarely needed
Cannot leave all genes on all the time – takes energy for transcription and translation – drain cell of energy
Scientists – use Operon system

3. The lac Operon The lac operon - first operon discovered
Contains 3 genes coding for E. coli proteins that permit the bacteria to use the sugar lactose
Galactoside permease (lacZ) - transports lactose into the cells
b-galactosidase (lacY) cuts the lactose into galactose and glucose
Galactoside transacetylase (lacA)- function is unclear
In a flask containing glucose and lactose, the cells exhaust glucose and stop growing. For a short time it appears that it cannot adjust to lactose, but resumes growth after some time and turn on their lac operon to begin to accumulate their enzymes needed to metabolize lactose.
Lactose is composed of two simple sugars galactose and glucose joined by beta galactosidic bond.

4. lac Operon
All 3 genes are transcribed together producing 1 mRNA - a polycistronic message that starts from a single promoter
Cistron or gene - has its own ribosome binding site
A polycistronic message with information from more than one gene. Each cistron can be translated by separate ribosomes that bind independently of each other

5. Control of the lac Operon
The lac operon is tightly controlled - using 2 types of control
Negative control - like the brake of a car - binds the repressor to the operator
Positive control – like accelerator pedal – removes repressor from operator
Activator - additional positive factor - responds to low glucose by stimulating transcription of the lac operon
The brake in negative control is a protein called lac repressor which keeps operon turned off or repressed as long as lactose is absent. Because it would be wasteful for the cell to produce enzymes needed to use an absent sugar. An additional factor activator is needed to activate operon. It responds to low glucose levels by stimulating transcription of lac operon, but high glucose levels keeps concentration of activator low so transcription of operon cannot be stimulated. The advantage of this positive control is that it keeps operon turned off when level of glucose is high as bacteria metabolize glucose more easily than lactose and it will be wasteful for them to activate the lac operon in presence of glucose

6. Negative Control of the lac Operon

7. Negative Control of the lac Operon
Negative control - operon is turned on unless something turns it off and stops it
The off-regulation is done by the lac repressor
Product of the lacI gene
Tetramer of 4 identical polypeptides
Binds the operator just right of promoter
Repressor is the protein that turns operon off. When repressor binds the operator, operon is repressed. The operator and promoter are contiguous. When repressor bind to operator then it prevents RNA polymerase from binding to the promoter and transcribing the operon.
As long as no lactose is available, lac operon is repressed

8. lac repressor When repressor binds the operator - operon is repressed
Operator and promoter are contiguous
Repressor bound to operator prevents RNA polymerase from binding to the promoter
As long as no lactose is available - lac operon is repressed

9. Inducer of lac operon The repressor is an allosteric protein
Binding of one molecule (inducer – allolactose) to the protein (repressor) changes shape of a remote site on that protein
Altering its interaction with a second molecule (operator)
Inducer (one molecule) of lac operon binds the repressor causing the repressor to change conformation that favors release from the operator (the second molecule). The inducer is allolactose, an alternative form of lactose. This happens in absence of lactose

10. Mechanism Summary
Two hypotheses of mechanism for repression of the lac operon
RNA polymerase can bind to lac promoter in presence of repressor
Repressor will inhibit transition from abortive transcription to processive transcription
Repressor by binding to operator blocks access by the polymerase to adjacent promoter

11. Three lac operators Three lac operators
The major lac operator lies adjacent to promoter
Two auxiliary lac operators - one upstream and the other downstream
All three operators are required for optimum repression
The major operator produces only a modest amount of repression

12. Role in repression of transcription by the three operators

13. Catabolite repression
When glucose is present - lac operon is in an inactive state
Selection in favor of glucose attributed to role of a breakdown product – Catabolite
Catabolite repression uses catabolite to repress the operon
Selection in favor of glucose attributed to role of a breakdown product – Catabolite. The breakdown product is related to glucose or catabolite of glucose. This happens when both glucose and lactose are present. The bacteria will not use lactose until it metabolizes glucose

14. Positive Control of the lac Operon
Positive control of lac operon by a substance sensing lack of glucose -activate lac promoter
The conc. of nucleotide - cyclic-AMP - rises as the concentration of glucose drops

15. Catabolite Activator Protein
cAMP added to E. coli can overcome catabolite repression of lac operon
Positive controller of lac operon has 2 parts:
cAMP + protein factor
Protein factor is known as:
Catabolite activator protein or CAP
Cyclic-AMP receptor protein or CRP
Gene encoding this protein is crp
Addition of cAMP led to activation of the lac gene even in the presence of glucose

16. The Mechanism of CAP Action
CAP-cAMP complex binds to the lac promoter
Mutants whose lac gene is not stimulated by complex had the mutation in the lac promoter
Mapping the DNA has shown that the activator-binding site lies just upstream of the promoter
Binding of CAP and cAMP to the activator site helps RNA polymerase form an open promoter complex

17. CAP-cAMP complex
The open promoter complex does not form even if RNA polymerase has bound to the DNA - till the CAP-cAMP complex is also bound
CAP-cAMP recruits polymerase to the promoter in two steps
Formation of the closed promoter complex
Conversion of the closed promoter complex into the open promoter complex

18. CAP
Binding sites for CAP in lac, gal and ara operons -contain the sequence TGTGA
Binding of CAP-cAMP complex to DNA is tight
CAP-cAMP activated operons have very weak promoters
Their -35 boxes are quite unlike the consensus sequence
Sequence conservation suggests an important role in CAP binding

19. CAP-cAMP Activation of lac Transcription
The CAP-cAMP dimer binds to its target site on the DNA
The aCTD (a-carboxy terminal domain) of polymerase interacts with a specific site on CAP
Binding is strengthened between promoter and polymerase

20. ara Operon
The AraC - ara control protein, acts as both a positive and negative regulator
There are 3 binding sites
Far upstream site, araO2
araO1 located between -106 and -144
araI is really 2 half-sites
araI1 between -56 and -78
araI to -51

21. araCBAD operon
The ara operon is also called the araCBAD operon for its 4 genes
Three genes, araB, A, and D, encode the arabinose metabolizing enzymes
These are transcribed rightward from the promoter araPBAD
Other gene, araC
Encodes the control protein AraC
Transcribed leftward from the araPc promoter

22. Control of the ara operon

23. AraC Control of the ara Operon
In absence of arabinose - no araBAD products - AraC exerts negative control
Binds to araO2 and araI1
Loops out the DNA in between
Represses the operon
Presence of arabinose - AraC changes conformation
It can no longer bind to araO2
Occupies araI1 and araI2 instead
Repression loop broken
Operon is derepressed

24. Positive Control of the ara Operon
Positive control is also mediated by CAP and cAMP
The CAP-cAMP complex attaches to its binding site upstream of the araBAD promoter

25. The trp Operon
The E. coli trp operon contains the genes for the enzymes - to make the amino acid tryptophan
The trp operon codes for anabolic enzymes
Anabolic enzymes are typically turned off by a high level of the substance produced
Operon - negative control by a repressor - tryptophan levels are elevated
trp operon also exhibits attenuation
anabolic enzymes those that build up a substance. This operon is subject to negative control by a repressor when tryptophan levels are elevated

26. Tryptophan’s Role in Negative Control of the trp Operon
Five genes code for the polypeptides in the enzymes of tryptophan synthesis – Chorismic acid
The trp operator lies wholly within the trp promoter
Presence of high tryptophan helps the trp repressor bind to its operator
In lac operon, the promoter and operator precede the genes and same is true for trp operon. In trp operon the operator lies within the promoter. In positive control of lac operon, the cell senses the presence of lactose by appearance of tiny amounts of its rearranged product – allolactose. This causes the repressor to fall off lac operator and derepress the operon.

27. Negative control of trp operon
Without tryptophan no trp repressor exists -inactive protein - aporepressor
If aporepressor + tryptophan - changes conformation with high affinity for trp operator
Combine aporepressor and tryptophan to have the trp repressor
Tryptophan is a corepressor

28. Mechanism of Attenuation
Attenuation imposes an extra level of control on an operon - more than just the repressor-operator system
Operates by causing premature termination of the operon’s transcript when product is abundant
The reason for premature termination is attenuator contains a transcription stop signal (terminator): an inverted repeat followed by a string of eightA-T pairs in a row. Because of inverted repeat the transcript of this region would tend to engage in intramolecular base pairing forming hairpin.

29. Defeating Attenuation
Attenuation operates in the E. coli trp operon as long as tryptophan is plentiful
If amino acid supply low - ribosomes stall at the tandem tryptophan codons in the trp leader
trp leader being synthesized as stalling occurs - stalled ribosome will influence the way RNA folds
Prevents formation of a hairpin

30. Overriding Attenuation

31. Riboswitches
Small molecules can act directly on the 5’-UTRs of mRNAs to control their expression
Regions of 5’-UTRs capable of altering their structures to control gene expression in response to ligand binding are called riboswitches
Region that binds to the ligand is an aptamer

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