Conversion of LuxR

1
Conversion of LuxR
LuxR
Transcription
Some Scientific Tinkering
LuxR
Transcription
2
How?
RNA Polymerase Binding Site Consensus Sequences
Transcriptional Start Site
20 bp lux box
bp
-42.5
-35
0
-10
RNA Polymerase Binding Site Consensus Sequences
Transcriptional Start Site
20 bp lux box
bp
-42.5
-35
0
-10
3
What is LuxR / lux?
Vibrio fischeri a bioluminescent marine bacteria
The BIOLUMINESCENCE comes from the lux operon.
LuxR is a transcriptional activator of the lux
operon that is activated in dense cell
environments. Lux boxes bind LuxR and RNA
polymerase to help initiate transcription.
SO,
Accumulation of 3-oxo-C6 HSL
High Cell Density
LuxR Activated
LuxR interacts with RNAP and binds to lux box
Transcription of bioluminescent genes start
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In this experiment
b - galactosidase
RNA Polymerase Binding Site Consensus Sequences
lacZ Start Site
20 bp lux box
bp
-42.5
0
-35
-10
b - galactosidase
RNA Polymerase Binding Site Consensus Sequences
lacZ Start Site
lac sequence
bp
-42.5
0
-35
-10
5
Results (Fig. 2)!

• Found that with the experimental plasmid,
  b-galactosidase production stopped when
  3-oxo-C6-HSL was added (this usually activated
  LuxR, but it appears to be repressed)
• With control lacZ plasmid, b-galactosidase
  production occurred with or without 3-oxo-C6-HSL

6
Results (Fig. 3)!!

• The higher the concentration of 3-oxo-C6-HSL, the
  greater the repression by the lux box plasmid.
• The higher the concentration of a similar
  compound, C8-HSL, the greater the repression
• This is the OPPOSITE of what would happen normally

7
Results (Fig. 4)!!

• LuxR represses lacZ even when its downstream of
  the promoter

b - galactosidase
RNA Polymerase Binding Site Consensus Sequences
lacZ Start Site
lux box
bp
-42.5
-35
0
-10
8
Results (Fig. 5)!!

• Adding a lot of lux boxes elsewhere made
  repression of lacZ less efficient, showing that
  repression is actually caused by LuxR binding to
  the lux boxes

Where should I bind?
9
Results (Fig. 6)!!

• Analysis of mutant LuxRs
• LuxR has an N-terminus that either binds to the
  signal OR binds to the C-terminus
• LuxR has a C-terminus which is bound to RNAP and
  lux box OR the N-terminus

10
Results (Fig. 6)!!

• C-terminal deletions resulted in an inability to
  bind to RNAP
• Normally, this lessens the transcription level
• In this experiment, it increases transcription
  level (lowers repression level)
• N-terminal deletions of 5 or 10 AAs resulted in
  signal-dependent repressors
• N-terminal deletions of 20 and 127 AAs resulted
  in loss of repression ability

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Results (Table 2)!!

• Which came first, the chicken or the egg?
• Rephrased Which came first, signal binding or
  DNA binding?
• Hopefully obvious to synthetic biologists, signal
  binding comes first
• Looked at single AA substitutions in LuxR to
  determine this

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Conclusions

• LuxR acts as a repressor when the lux box is in
  between the RNAP binding consensus sequences
• LuxR needs both its N-terminal and C-terminal
  ends to function as a repressor