BacGen Exam III: Positive Regulation, Attenuation, and Feedback Inhibition

Explain how hairpins are form in the trpL region?

1. After RNA polymerase initiates transcription at the promoter, it moves through the trpL region to a site located just after region 2, where it pauses. 2. The hairpin formed by region 1 and region 2 is the signal to pause 3. The pause if very brief (1 second) but it gives a ribosome time to load onto the mRNA before the RNA polymerase proceeds to region 3. 4. Ribosome helps release the paused RNA polymerase by colliding with it 5. The progress of the ribosome through the trp codons of the trpL determines whether hairpin 3:4 will form, causing termination, or whether region 2 will instead pair with region 3 6. `The 2:3 hairpin will form if the ribosome stalls at the trp codons because of low tryptophan concentrations

What did early evidence indicate about the lac and the ara operons? explain.

1. The loss of the operon regulors resulted in very different phenotypes. Deletions and nonsense mutantions in the araC gene showed a supperrepressed phenotype, where the genes were not express even in the presence oft inducer arabinose. While mutations in the regulator for the negatively regulated lac operon resulted in constitutive phenotypes. 2. The frequency of constitutive mutants Constitutive mutants are common in a negatively regulated operon, such as lac, but mutants that constitutively express the ara operon were extremely rare. Taken together this evidence suggested that the ara operon was regulated by a mechanism other the negative regulation.

why araCC mutations are recessive to wild type?

AraCC in the P2 form can't bind araI1 and araI2 if the wild-type AraC is already bound to araO2 and araI1. The DNA is already bent

What role do attenuation play during transcription? give an example of attenuation

Attenuation allows transcription to begin at the promoter but terminates it before the RNA polymerase reaches the first structural gene and occurs only if the products of the operon are not needed. Example: the E.coli trp operon

other than the Pbad promoter what else is just upstream of the aral site?

CAP site where CAP binds

If we didn't know araC had dual states/functions what would we expect the phenotype of a mutant araC to be?

Constitutive araC mutantations are predicted to be dominant over the wild-type allele in complementation tests. If araC protein is only an activator, partial dipolid cells with the constitutive araC allele and the wild type allele would be expected to constitutively express the ara operon.

How might you identify feedback inhibition mutations of the Isoleucine-Valine Operon?

If E. coli cells are presented with high concentrations of valine they will die as long as isoleucine is not provided in the medium. Valine and isoleucine are synthesized by the same pathway encoded by the ilv operon. The first enzyme of the pathway is feedback inhibited by valine, so the cells make neither valine or isoleucine The cells starve for isoleucine unless it is provided in the medium

When will the 3: 4 hairpin form?

If the ribosome does not stall at the trp codons, it will continue until it reaches the UGA stop codon at the end of the trpL. It remains there while region 4 is synthesized and it prevents formation of hairpin 2:3 Transcription will terminate

How might you identify feedback inhibition mutations in the trp operon?

Plate on 5-methyltrpophan Only mutants defective in feedback inhibition can multiply to form a colony in the absence of tryptophan(and 5-methyl-tryptophan . This is due to the mutation in the trpE gene that prevents the binding of tryptophan to anthranilate synthetase enzyme

How can you isolate constitutive mutants of the ara operon?

Plate on media containing the ara anti-inducer D-fucose D-fucose binds to the AraC protein and prevents it from binding to L-arabinose thus preventing ara operon induction. Wild-type E.coli can't form colonies on D-fucose plate with L-arabinose as the sole carbons source. Therefore, only mutants which constitutively express the genes of the ara operon can form colonies under these conditions.

What is the role of the trpL leader region in attuation of the trp operon? what experimental evidence suggests this explanation?

Regulation of the trp operon is targeted by not the promoter but the trpL region. This is shown when deletion of the leader region eliminates regulation by attenuation Double mutants with deletions of trpL and a trpR mutation are completely constitutive for expression of the trp operon. Deletions of trpL were found to be cis-acting affecting only expression of the trp operon on the same DNA. Transcription of the operon was found to terminate in the trpL region in the presence of tryptophan because of excess tRNATrp

In what two state can the AraC protein exist and explain each state.

The AraC protein can exist in two states: P1 and P2. AraC is inactive in the P1 state in the absence of the inducer, L-arbinose. AraC is active in the P2 state in the presence of L-arabnose.

what is AraC? what results from it exerting its function and what kind of protein is AraC

The activator protein AraC binds to a site upstream of the promoter called the araI region to activate transcription in the presence of the inducer arabinose

what promoter is AraC transcribed from and where is this promoter located?

The araC gene is transcribed from a promoter pC in the opposite direction from pBAD

True or False: AraC can regulate its own expression. Explain why

True When arabinose isn't present, AraC are bound at araO2 and araI1 bending the DNA in the region of the araC promoter pC .If the concentration of AraC becomes too high it binds to the operator araO1 preventing transcription from pC

What explains why mutations in araC that cause the constitutive phenotype are rare.

When L-arabinose is present AraC binds to araL in the promoter it activates transcription of the ara operon. araCC mutations change AraC so that it is permanently in the P2 state even in the absence of L-arabinose, so it is a constitutive mutant. These mutations are very rare since only a few amino acid changes in the AraC protein could change the conformation to the P2 state

What is a positively regulated operon? give an example

an operon that is under the control of an activator protein. The genes of the operon are only transcribed in the presence of activator when the inducer is bound. Example: the L-ara operon

name the structural genes of the L-ara operon. What promoter are these genes transcribed from?

araA, araB, and araC which are transcribed from a single promoter pBAD

feedback inhibition is common in

bio-synthetic pathways

What does formation of a 3:4 hairpin cause?

causes RNA polymerase to terminate transcription because this hairpin is part of a factor-independent transcription termination signal

When will the 2:3 hairpin form?

if the ribosome stalls at the trp codons because of low tryptophan concentrations Transcription will continue

how could you prove that araC has dual states? What do we know to be true?

introduce an F' factor with a wild type ara operon in a cell with an araC constitutive mutant allele If araC is solely an activator protein the partial merodipolid cells that have both the araC constitutive mutant allele and the wild type allele are predicted to constitutive express the arabinose operon. There all the ara genes will be constitutive. The araCC Mutation is actually Recessive.

What is araC in its P2 form? What is its function?

its an activator In the presence of arabinose the AraC protein changes to the P2 state and now preferentially binds to araI1 and araI2 activating the operon.

What is araC in its P1 form? What is its function?

its an anti-activator The AraC P1 state binds to the operator araO2 and another site araI1 bending the DNA. When bound to araO2 it cannot bind to araI2 and activate transcription from the pBAD promoter

What evidence is there for the exitance and function of the 2:3 hair pain?

phenotypes produced by mutation trpL75 This mutation changes one nucleotide and prevents formation of the 2:3 hairpin. In trpL75 mutants transcription terminates in the trpL region even in the absence of tryptophan

When the ribosome encounters the trp codon under low tryptophan conditions it...

stalls in the trpL region

the stall ribosome in the trpL region signals....

that tryptophan concentration is low and transcription should continue

What is feedback inhibition?

the end product of the pathway binds to the first enzyme of the pathway inhibiting its activity. Its a more sensitive and rapid mechanism for modulating the amount of the end product than are transcriptional regulation and translational regulation. The trp operon uses feedback inhibition

Hairpin formation in the trp operon is determined by...

the levels of aminoacylated tRNATrp

If TrpR was the only way to regulate expression of the trp operon, then levels of the enzymes in a TrpR mutant should always be the same whether tryptophan is present or absent. However, in a trpR null mutant, the expression of the enzymes is higher when tryptophan is absent than when its present. Why is this the case? (Hint: tRNA-Trp)

trpR is likely not the only regulator in the trp operon. It was found that mutations in the tryptophanyl-tRNA synthetase, the structural gene for the tRNA-Trp, other genes whose products modify tRNA-Trp all increase expression in the operon in this situation. All of these mutatations resulted in lower expression levels of tRNA-Trp. Therefore the operon is not sensing the concentration of tryptophan in the cell, but rather the levels of tryptophan bound to the tRNA

Where does AraC bind to repress transcription?

two operators, araO1 and araO2

Trp Operon Attenuation is based on...

which of several secondary structure hairpins forms in the leader RNA.

If levels of tryptophan are low the levels of tRNA-Trp..

will also be low

Four different regions in the trpL leader RNA, regions 1, 2, 3, and 4 can form three different hairpins, where are these hairpins?

•1:2, 2:3, and 3:4