Lecture 6 Flashcards Preview

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Flashcards in Lecture 6 Deck (25):
1

DeltaED53 and LD102 mutation in the Trp operon effect:

- Different mechanisms due to the differing ratio's of expression when a TrpR+ is compared to a trpR- mutation.
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2

DeltaLD102 mutation is due to a...

- 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.

3

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

- 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.

4

What is TrpT?

- It codes for a tRNA molecule with the trp anticodon.
- It is used to translated trp codons

5

What is TrpX?

- An enzyme that adds a small modification to the tRNAtrp
- This enzyme is needed to make trp functional

6

What is TrpS?

- It codes for tryptophanyl-tRNAtrp synthetase
- It attaches tryptophan to modified tRNAtrp

7

What are TrpRX and S involved in?

- Producing active tryptophanyltRNAtrp
- Used for translation by ribosomes

8

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

- 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.

9

What does the Trp leader region mRNA form?

- 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.

10

What happens when there is high tryptophan?

- 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

11

What happens when there is low tryptophan?

- 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.

12

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

- The ribosome!
- It is determining the outcome of this mechanism

13

What is the effector for trp attenuation?

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

14

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

- 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.

15

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

- High tryptophan: fully repressed, most transcripts terminate
- Low tryptophan: fully induced, most transcripts don't terminate.

16

Attenuation regulates a number of amino acid biosynthetic genes including:

- 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.

17

Regulation of the tryptophan operon in B. subtilis

- 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.

18

What is a TRAP termination mechanism?

- 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!

19

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

- Tryptophan

20

Is the TRAP mechanism positive or negative?

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

21

Is the TRAP mechanism inducible or repressible?

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

22

What is anti-TRAP?

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

23

How is antiTRAP influenced?

- 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.

24

E.coli:

Two independent mechanisms, two effectors,
- TrpR and tryptophan: negative repressible AND
- Ribosome + tryptophanyl-rTHANtrp

25

B.subtilis:

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