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LuxR Transcriptional Activation: Mechanisms & Mutations in Vibrio Fischeri

Learn about LuxR's role in Lux operon transcription, its interactions with RNA polymerase, and the impact of mutations on gene repression and activation in bioluminescent bacteria. Discover how LuxR functions as a repressor and the importance of its N-terminal and C-terminal ends in gene regulation.

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LuxR Transcriptional Activation: Mechanisms & Mutations in Vibrio Fischeri

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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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