Lecture 6 Flashcards
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.
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.
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.
What is TrpT?
- It codes for a tRNA molecule with the trp anticodon.
- It is used to translated trp codons
What is TrpX?
- An enzyme that adds a small modification to the tRNAtrp
- This enzyme is needed to make trp functional
What is TrpS?
- It codes for tryptophanyl-tRNAtrp synthetase
- It attaches tryptophan to modified tRNAtrp
What are TrpRX and S involved in?
- Producing active tryptophanyltRNAtrp
- Used for translation by ribosomes
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.
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.
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
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.
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
What is the effector for trp attenuation?
- charged tRNAtrp, which is influenced by the level of tryptophan.
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.
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.
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.
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.
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!
What is the small MW effector molecule in the TRAP tryptophan mechanism?
- Tryptophan
Is the TRAP mechanism positive or negative?
- A negative system because if TRAP was absent expression would always occur
Is the TRAP mechanism inducible or repressible?
- Repressible as it is acting to bring levels of trp down.
What is anti-TRAP?
- A gene encoding antiTRAP which binds to and inactivates TRAP.
- If there is lots of antiTRAP it will bind TRAP
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.
E.coli:
Two independent mechanisms, two effectors,
- TrpR and tryptophan: negative repressible AND
- Ribosome + tryptophanyl-rTHANtrp
B.subtilis:
One mechanism: two effectors
- tRNAtrp: inducer of antiTRAP
and antiTRAP: negative regulator of TRAP
- TRAP + tryptophan: negative, repressible
