Regulation of Biosynthetic Operons

1
Regulation of Biosynthetic Operons

• Regulation of biosynthetic operons are opposite
  to degradative operons (e.g. lac).
• Enzymes should not be turned on when end product
  present.
• Model system is tryptophan biosynthesis operon
  (trp operon) of E. coli.
• Negative regulation if tryptophan is present,
  interacts with repressor to block promoter.
• Tryptophan is corepressor

2
trp Operon
Structural genes
Ptrp trpL trpE trpD trpC trpB trpA ter
trpR
Leader peptide
terminator
promoter
Aporepressor
Tryptophan
Anthranilic acid
Chorismic acid
trp operon encodes genes for tryptophan synthesis
3
When tryptophan is present, Operon turned off
Aporepressor
Structural genes
Ptrp trpL trpE trpD trpC trpB trpA ter
trpR
Repressor blocks promoter RNA pol cant bind No
transcription
tryptophan
Aporepressor
Tryptophan binds to aporepressor
4
When tryptophan is absent, Operon turned on
Aporepressor
Structural genes
Ptrp trpL trpE trpD trpC trpB trpA ter
trpR
RNA polymerase binds promoter, Transcription
initiated
Aporepressor can not bind to promoter
unless tryptophan is present.
5
trpR is Autoregulated

• TrpR negatively regulates trp operon in presence
  of tryptophan, and also its own transcription.
• When TrpR absent, 70x higher transcription.
• What advantage is autoregulation of repressor?

6
trpR blocks its own transcription when tryptophan
is present
Repressor
Structural genes
Ptrp trpL trpE trpD trpC trpB trpA ter
trpR
RNA polymerase binds promoter, Transcription
initiated
TrpR not needed Shuts down
Aporepressor can not bind to promoter
7
tnpR derepressed when tryptophan present
aporepressor
Structural genes
Ptrp trpL trpE trpD trpC trpB trpA ter
trpR
tryptophan
aporepressor
Most trpR combines with tryptophan
Tryptophan binds to aporepressor
8
Regulation by Attenuation

• Attenuation terminates transcription before
  transcription of first structural gene.
• Attenuation has a 10x effect (700x total).
• trpL encodes a leader sequence
• Encodes two adjacent tryptophan codons
• If tryptophan is low, levels of
  tryptophan-tRNAtrp also low
• Ribosome will stall because cant find
  tryptophan-tRNAtrp
• Transcription should continue.

9

• Leader (trpL) mRNA can form three different
  hairpin loops.
• Alternative mRNA structures determine which loops
  form.
• One of the hairpin loops is factor independent
  terminator.

10
Leader peptide (trpL) structure
Hairpin loop regions
4
2
3
1
Promoter
ATG TT TGA
Transcription start
Attenuator
Leader peptide coding region
Structural genes
Pause site
TT 2 trp codons
NOTE the attenuator is a special terminator.
11
Attenuation using translation to sense amino
acid levels
Tryptophan abundance
Tryptophan starvation
2
3
transcription continues
transcription terminates
4
ribosome falls off at terminator
12
Termination Conformation
Ribosome Stop codon
Pause structure
A
Terminator hairpin
G
U
T T
Region 2
Region 1
Region 3
Region 4
AUG
UUUUUUU
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Readthrough Conformation
Antiterminator Structure
Note the single hairpin structure is less stable
than the 2 hairpins.
Region 3
Region 2
AUG
UUUUUUUU
Region 1
Region 4
If Regions 2 and 3 form hairpin loop, 12 and
34 can not form hairpins.
14
Polymerase Pauses at Pause Site
(allows ribosome to attach)
TT 2 trp codons
4
2
3
1
Promoter
ATG TT TGA
Transcription start
2
Pause site
1
mRNA
15
Pause Allows Ribosomes to Load
TT 2 trp codons
4
2
3
1
Promoter
ATG TT TGA
Transcription start
1
2
Pause site
Ribosome
16
If sufficient trp-tRNAtrp , ribosome doesnt
stall.
TT 2 trp codons
Ribosome stop codon
4
2
3
1
Promoter
ATG TT TGA
Transcription start
3
4
2
1
Ribosome interferes with 23 hairpin
Termination
Ribosome terminates at UGA stop
17
If sufficient trp-tRNAtrp , Ribosomes Dont
Stall.
TT 2 trp codons
4
2
3
1
Promoter
ATG TT TGA
Transcription start
3
4
2
1
Ribosome interferes with 23 hairpin
Termination
May even knock RNA pol off
18
If Insufficient trp-tRNAtrp , Ribosomes Stall at
TT.
TT 2 trp codons
Ribosome cant find trp-tRNAtrp
4
2
3
1
Promoter
ATG TT TGA
1
Transcription start
4
TT
1
2
Terminator 34 cant form
3
2
Note 1 2 can not hairpin.
Ribosome
Transcription proceeds