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Conversion of LuxR

Conversion of LuxR. LuxR. Transcription. Some Scientific Tinkering…. LuxR. Transcription. 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.

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Conversion of LuxR

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  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

  4. In this experiment… b - galactosidase RNA Polymerase Binding Site Consensus Sequences lacZ Start Site 20 bp lux box bp -42.5 -35 0 -10 b - galactosidase RNA Polymerase Binding Site Consensus Sequences lacZ Start Site lac sequence bp -42.5 -35 0 -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 it’s downstream of the promoter b - galactosidase RNA Polymerase Binding Site Consensus Sequences lacZ Start Site lux box bp -42.5 -35 -10 0

  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 LuxR’s • 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 AA’s resulted in signal-dependent repressors • N-terminal deletions of 20 and 127 AA’s resulted in loss of repression ability

  11. 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

  12. 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

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