Tryptophan operon leader sequence regulation in E coli and B subtilis

Question 1

What does the Trp operon look like in E coli?

Answer 1

Polycistronic mRNA that has 5 opening reading frames. Each one encodes an enzyme used in Trp biosynthesis

Question 2

What are the 3 mechanisms by which the Trp operon in E coli is regulated?

Answer 2

1. Feedback inhibition
2. TrpR repressor
3. Leader sequence mediated transcription attenuation

Question 3

How does feedback inhibition regulate the Trp operon in E coli?

Answer 3

Trp will bind to the first enzyme in the biosynthesis pathway (anthranilate synthetase) and inhibits it, blocking more Trp from being made

Question 4

Why isn’t feedback inhibition sufficient to suppress the Trp operon in E coli?

Answer 4

It only blocks enzyme activity and doesn’t stop more enzymes from being made. The cell is still wasting energy making proteins it doesn’t need

Question 5

How does the TrpR repressor work?

Answer 5

If Trp levels are low, TrpR is in a conformation that prevents it from binding to DNA so it doesn’t block transcription. If Trp levels are high, it will bind to TrpR and change its conformation so that it binds to the promoter and physically blocks RNAP from binding

Question 6

Why isn’t the TrpR repressor sufficient to suppress the Trp operon in E coli?

Answer 6

Leaky repression. Some expression will still be going through

Question 7

How many different hairpin structures can the leader sequence in the Trp operon in E coli form?

Answer 7

3. The pause hairpin (regions 1 and 2), the terminator hairpin (regions 3+4), and the anti-terminator hairpin (regions 2+3)

Question 8

Which hairpin will form first when RNAP starts transcribing the Trp operon?

Answer 8

The pause hairpin

Question 9

What are the two options that can occur after the pause hairpin causes RNAP to stop transcribing the Trp operon in E coli?

Answer 9

1. The terminator hairpin forms and transcription stops

2. The pause hairpin unwinds and the anti-terminator hairpin forms, and transcription continues

Question 10

Which secondary structure of leader sequence in the Trp operon in E coli is more energetically favourable?

Answer 10

The pause + terminator hairpin

Question 11

What allows the anti-terminator hairpin to form when its needed in the Trp operon in E coli?

Answer 11

Coupled translation of the leader peptide

Question 12

What is the leader peptide?

Answer 12

A tiny peptide encoded by a tiny ORF in the leader sequence. It has no function and is useless itself

Question 13

If the leader peptide has no function itself, why is it so important to the regulation of the Trp operon in E coli?

Answer 13

It contains two codons for Trp. If the ribosome can’t put those in, then it can’t finish translating the peptide and the terminator hairpin can’t form

Question 14

What is the sequence of events that leads to expression of the E coli Trp operon when Trp levels in the cell are low?

Answer 14

1. RNAP binds to the promoter and starts transcribing
2. It transcribes the pause hairpin, which will form and cause RNAP to stop
3. A ribosome will bind to the transcript and starting translating the leader peptide
4. It reaches the Trp codons, and can’t put them in, so it pauses
5. The pause hairpin unravels and the anti-terminator hairpin forms
6. Transcription proceeds and the Trp operon is expressed

Question 15

What is the sequence of events that leads to expression of the E coli Trp operon when Trp levels in the cell are high?

Answer 15

1. RNAP binds to the promoter and starts transcribing
2. It transcribes the pause hairpin, and RNAP stops
3. A ribosome binds to the transcript and starts translating the leader peptide
4. There’s lot of Trp, so the ribosome just adds it in and finishes translating the peptide
5. It falls off, and the terminator hairpin forms
6. RNAP dissociates from the operon and transcription is attenuated

Question 16

Do RNA hairpins affect translation?

Answer 16

No, ribosomes have helicase activity and just unwind them and go

Question 17

Does the mechanism of leader sequence based regulation used in the E coli Trp operon work in eukaryotes?

Answer 17

No, transcription and translation aren’t coupled

Question 18

What are the 3 similarities between the regulation of the Trp operon in E coli and in B subtilis?

Answer 18

1. Respond to levels of Trp in the cell
2. Use terminator and anti-terminator hairpins
3. Use attenuation to stop expression of the operon under high Trp conditions

Question 19

What are the 3 differences between the regulation of the Trp operon in E coli and in B subtilis?

Answer 19

1. B subtilis uses an RNA binding protein called TRAP, which only binds to RNA when it is bound to Trp
2. B subtilis uses this process to attenuate transcription and suppress translation
3. The anti-terminator hairpin is more stable

Question 20

What is the sequence of events that leads to expression of the B subtilis Trp operon when Trp levels in the cell are low?

Answer 20

1. RNAP binds to the promoter and starts transcribing the operon
2. TRAP isn’t binding to Trp and stays inactive
3. The anti-terminator hairpin forms spontaneously
4. Transcription continues

Question 21

What is the sequence of events that leads to expression of the B subtilis Trp operon when Trp levels in the cell are high?

Answer 21

1. RNAP binds to the promoter and starts transcribing the operon
2. TRAP is binding to Trp, and it binds to the leader sequences in RNA transcript
3. The anti-terminator hairpin can’t form, and the terminator hairpin forms instead
4. Transcription is attenuated

Question 22

How does B subtilis ensure the Trp operon is completely shut off when Trp levels are high?

Answer 22

A second hairpin forms later in the transcript that sequesters the ribosome binding site. So even if some transcription goes through, it won’t be translated

Question 23

Why doesn’t the second hairpin form later in the transcript of the B subtilis Trp operon when the anti-terminator hairpin is present?

Answer 23

The anti-terminator locks up the sequences needed for that second hairpin to form, which prevents the ribosome binding site from being sequestered