Lecture 6

Question 1

DeltaED53 and LD102 mutation in the Trp operon effect:

Answer 1

• ## Different mechanisms due to the differing ratio’s of expression when a TrpR+ is compared to a trpR- mutation.

Question 2

DeltaLD102 mutation is due to a…

Answer 2

• Cis acting gmutation that deletes a terminator in the TrpR causing an increase in expression.
• Downstream of the promoter, but upstream of the structural genes.

Question 3

Trans acting mutations in TrpT, TrpX and TrpS cause ___ for the lac operon

Answer 3

• An increase expression of the lac operon.
• They have no effect in the deltaLD102 attenuator deletion mutant, indication they affect the same regulatory mechanism (attenuation).
• They behave independently of TrpR, but they act together.

Question 4

What is TrpT?

Answer 4

• It codes for a tRNA molecule with the trp anticodon.

- It is used to translated trp codons

Question 5

What is TrpX?

Answer 5

• An enzyme that adds a small modification to the tRNAtrp

- This enzyme is needed to make trp functional

Question 6

What is TrpS?

Answer 6

• It codes for tryptophanyl-tRNAtrp synthetase

- It attaches tryptophan to modified tRNAtrp

Question 7

What are TrpRX and S involved in?

Answer 7

• Producing active tryptophanyltRNAtrp

- Used for translation by ribosomes

Question 8

What does the leader region of the Trp mRNA code for? Attenuation mechanism in E.coli:

Answer 8

• A short ‘leader peptide’ containing potential stem-and-loop structures.
• These can act as a terminator based on the position of the ribosome over the base pairing regions.
• The position of the ribosome is determined by the level of tRNAtrp in the cell.

Question 9

What does the Trp leader region mRNA form?

Answer 9

• Two mutually exclusive stemp-and-loop structures based on the
• Regions 3&4 pair (the factor-independant transcription terminator) to form the hairpin loop, and regions 1&2 pair.
  OR
• Regions 2&3 pair, and the terminator region is single-stranded, not causing termination.

Question 10

What happens when there is high tryptophan?

Answer 10

• The ribosome translates through to the leader peptide sequence and covering regions 1&2, preventing region 2 from pairing with region 3, allowing region 3 to pair with region 4 causing transcription (RNAP) termination

Question 11

What happens when there is low tryptophan?

Answer 11

• The ribosome pauses at the trp codons while translating the leader peptide sequence, due to the trp sequences to come along (there aren’t many of them).
• RNAP continues on, region 2 pairs with region 3, preventing 3 from pairing with 4. Transcription termination does not occur.
• Trp operon is transcribed by RNAP.

Question 12

What is acting as the small molecular weight effector molecule for trp attenuation/trans-acting regulatory ‘protein’?

Answer 12

• The ribosome!

- It is determining the outcome of this mechanism

Question 13

What is the effector for trp attenuation?

Answer 13

• charged tRNAtrp, which is influenced by the level of tryptophan.

Question 14

It the ribosome a positive or negative regulator of the trp attenuation mechanism?

Answer 14

• It is a positive regulator (if we imagine the ribosome doesn’t exist, as regions 1&2 will always pair as will regions 3&4.

Question 15

The tryptophan operon regulatory mechanisms respond to different levels of tryptophan:

Answer 15

• High tryptophan: fully repressed, most transcripts terminate
• Low tryptophan: fully induced, most transcripts don’t terminate.

Question 16

Attenuation regulates a number of amino acid biosynthetic genes including:

Answer 16

• Threonine, histidine, phenylalanine
• They each have a leader peptide sequence which is regulated by the level of the charged tRNA amino acid it encodes for.

Question 17

Regulation of the tryptophan operon in B. subtilis

Answer 17

• Leader region with two mutually exclusive base pairing stem and loop structures within the RNA.
• If A pairs with B, C can’t pair with D. If C pairs with D, a transcription terminator occurs in the form of a TRAP molecule.

Question 18

What is a TRAP termination mechanism?

Answer 18

• Trp RNA-binding attenuation protein (TRAP)
• TRAP binds tryptophan, not tRNAtrp. It forms an 11-mer complex wheel. Each TRAP monomer binds an AUG and a GAG sequence, forming a stable protein-RNA complex.
• Termination occurs!

Question 19

What is the small MW effector molecule in the TRAP tryptophan mechanism?

Answer 19

• Tryptophan

Question 20

Is the TRAP mechanism positive or negative?

Answer 20

• A negative system because if TRAP was absent expression would always occur

Question 21

Is the TRAP mechanism inducible or repressible?

Answer 21

• Repressible as it is acting to bring levels of trp down.

Question 22

What is anti-TRAP?

Answer 22

• A gene encoding antiTRAP which binds to and inactivates TRAP.
• If there is lots of antiTRAP it will bind TRAP

Question 23

How is antiTRAP influenced?

Answer 23

• The level of uncharged tRNAtrp
• If tryptophan is low uncharged tRNAtrp binds to antiTRAP mRNA, preventing formation of a terminator stem-and-loop, allowing antiTRAP expression.
• If trptophan is high there will be little uncharged tRNAtrp, so antiTRAP will not be expressed, TRAP is active and trp operon is expressed.

Question 24

E.coli:

Answer 24

Two independent mechanisms, two effectors,

• TrpR and tryptophan: negative repressible AND
• Ribosome + tryptophanyl-rTHANtrp

Question 25

B.subtilis:

Answer 25

One mechanism: two effectors
- tRNAtrp: inducer of antiTRAP
and antiTRAP: negative regulator of TRAP
- TRAP + tryptophan: negative, repressible