Green 15 - Trp Biosynthesis

Question 1

What 2 ways are there to regulate Trp biosynthesis in E. coli?

Answer 1

• repression of transcription via TrpR repressor protein

- translation efficiency of the leader peptide potentially leading to premature transcription termination

Question 2

How does the binding of Trp to the repressor protein affect its binding to the operator sequence and how?

Answer 2

TrpR homodimer binds to the Operator sequence of the Trp operon in the presence of Trp
Trp presence causes alignment of the DNA binding domains & recognition helices allowing them to bind in adjacent major grooves and block transcription

Question 3

What is the basis of Trp operon repression by transcriptional attenuation?

Answer 3

Transcription and translation are coupled together in bacteria

Question 4

Describe how the lack of Trp leads to the transcription of the trp genes in E. coli (based on transcriptional attenuation).

Answer 4

Leader sequence (within attenuator) contains 2 tandem Trp residues
Lack of Trp means that ribosomes cannot incorporate Trp into the leader peptide so the ribosome stalls here
This stalling allows the formation of a stem loop between 2:3 (antiterminator loop)
This allows the progression of RNAP and transcription of the Trp biosynthetic genes

Question 5

Within E. coli what is the purpose of the formation of the 1:2 antianti-terminator loop?

Answer 5

When mRNA initially produced 1:2 hairpin forms

This stalls the RNAP to enable a ribosome to bind @ the SDS and follow the RNAP in close proximity

Question 6

When both transcriptional and post transcriptional control mechanisms in E. coli are working, what is the overall increase in in expression of the Trp operon when Trp is present compared to not?

Answer 6

700 fold increase in Trp expression

Question 7

What are the 2 ways in which the B. subtillis Trp operon is regulated and controlled? (Here, the 5’ UTR of the Trp operon is switched between 2 conformations - terminator/anti)

Answer 7

TRAP and Anti-TRAP proteins

Question 8

What does TRAP stand for?

Answer 8

Trp-activated RNA-binding attenuation protein

Question 9

What are the 2 ways in which the expression of Anti-TRAP (AT) is controlled?

Answer 9

Tbox riboswitch

Leader peptide and translation efficiency

Question 10

Describe the Trp operon in B. subtillis.

Answer 10

Trp suboperon is contained within the arosupraoperon (for the synthesis of ALL aromatic aa)
The Trp suboperon has an UTR attenuator (attn) gene - for the formation of the TERM/ANTITERM stem loops
also has 6 genes encoding enzymes involved in Trp biosynthesis

Question 11

In the presence of Trp, how does TRAP stop the expression of the Trp operon?

Answer 11

TRAP binds to Trp
Allows TRAP to bind XAG repeats on attenuator antiterminator loop therefore stopping its formation, hence the terminator loop forms

Question 12

Give the structure of a TRAP protein.

Answer 12

Doughnut shaped
11 identical subunits
Can bind Trp between each subunit

Question 13

How does the presence of AT cause the expression of the Trp operon?

Answer 13

AT expressed under low levels of Trp
AT competes w/ XAG repeats for binding TRAP:Trp
Sequesters TRAP away from binding to XAGs and preventing formation of antiterminator
Antiterminator loop can form -> expression of Trp operon

Question 14

Describe the AT operon.

Answer 14

leader (5’ UTR)
rtpLP (leader peptide)
rtpA (AT protein)

Question 15

How is AT expression controlled based on the T box riboswitch?

Answer 15

low Trp = few loaded tRNAs w/ Trp therefore many unloaded
binds to the attenuator because no Trp there
makes 2 contacts w/ attenuator and stabilises formation of antiterminator loop
allows AT to be transcribed

Question 16

Describe the mode of operation of the leader peptide and how it influences AT expression.

Answer 16

Leader peptide contains 3 tandem Trp residues
Low Trp
- ribosome stalls @ W residues in the leader peptide sequence
- doesn’t block the SDS of the flanking rtpA gene therefore allows transcription of AT

High Trp

• ribosome doesnt stall and incorporates W in easily
• stops @ stop codon and blocks the SDS and start codon for rtpA therefore inaccessible to ribosomes
• no expression of rtpA