Sigma Factors & Transcriptional Regulation of P. syringae TTSS

1. Sigma Factors & Transcriptional Regulation of P. syringae TTSS Alexander Wong

2. Presentation Outline • RNApol holoenzyme • General properties of sigma factors • The alternative σ54 factor • Introduction to type III secretion system • Transcriptional regulation of Pseudomonas syringae TTSS • Conclusion

3. The RNApol holoenzyme • Definition of holoenzyme • Complete, working version of an enzyme • cf. apoenzyme - missing specific cofactors that allow it to perform its job • Examples of cofactors • common prosthetic groups (haem) or metal ions (magnesium) • Dissociable protein subunits – sigma (σ) factor.

4. The RNApol holoenzyme • All multi-subunit RNA polymerases have 5 core subunits. • Bacterial RNApol have additional σ subunit • Has function in binding to promoter • In bacteria, RNApol binds a promoter via σ • In eukaryotes, RNApol binds via TF complex • Bacterial RNApol is regulated purely by σ (initiation phase), but eukaryotic RNApol is regulated both by the TFs and by various gene regulatory proteins. • Although promoters are similar, the bacterial promoter tends to be highly conserved.

5. Presentation Outline • RNApol holoenzyme • General properties of sigma factors • The alternative σ54 factor • Introduction to type III secretion system • Transcriptional regulation of Pseudomonas syringae TTSS • Conclusion

6. General Properties of σ factor • RNA polymerase holoenzyme binds directly to DNA via its σ subunit • Promoter consensus sequence (below) is highly conserved in bacteria • Sequence alignment of E. coli promoters reveal a predominance of certain residues at positions -35 and – 10 relative to start point of transcription (+1). • Most common is the σ70 subunit – the generic sigma subunit

7. General Properties of σ factor

8. General Properties of σ factor • Bacteriophage-encoded σ factor also used to take over cellular transcriptional machinery

9. Presentation Outline • RNApol holoenzyme • General properties of sigma factors • The alternative σ54 factor • Introduction to type III secretion system • Transcriptional regulation of Pseudomonas syringae TTSS • Conclusion

10. The alternative σ54 factor • Most alternative sigmas are related in sequence and structure to σ70. • 2nd distinct type of σ called the σ54 family • Differences between the σ families • σ 54 family shares no sequence homology with the σ70 family • Whereas σ70 holoenzymes carry out this process of open complex formation on their own, σ 54 holoenyzmes require both an enhancer and ATP to perform this process.

11. The alternative σ54 factor • Activity of the alternative σ54 factor has been studied most intensively at the promoter for the glnA gene (encodes glutamine synthetase) • Closed complex → transcriptionally productive open complex requires the activator protein NTRC (aka. NRI) • Binds to sites with properties of eukaryotic transcriptional enhancers • NTRC must be phosphorylated, and this phosphorylation increases under nitrogen-limiting conditions

12. Presentation Outline • RNApol holoenzyme • General properties of sigma factors • The alternative σ54 factor • Introduction to type III secretion system • Transcriptional regulation of Pseudomonas syringae TTSS • Conclusion

13. Introduction to type III secretion system (TTSS) • System with many names – PEC, injectisome, TTSS, TTS etc. • Function to deliver bacterial proteins into target cells that then modulate host cell functions • Structural • Translocation • Effector proteins • Structurally homologous to bacterial flagellum • Genes usually clustered in mobile elements called pathogenicity islands (PAI) • Significance of research in bacterial pathogenicity and potential medical application

14. Introduction to type III secretion system (TTSS) Example: S. typhimurium TTSS1

15. hrp pathogenicity island • Shaded genes involved in regulatory functions • hrp box – promoter motif of HrpL • Expression of hrp genes induced by: • Pathogenesis • Acidic minimal salts medium

16. Presentation Outline • RNApol holoenzyme • General properties of sigma factors • The alternative σ54 factor • Introduction to type III secretion system • Transcriptional regulation of Pseudomonas syringae TTSS • Conclusion

17. Transcriptional regulation of Pseudomonas syringae TTSS HrpR HrpS HrpV ? pHrpL

18. Transcriptional regulation of Pseudomonas syringae TTSS • HrpR & HrpS forms heteromeric complex that functions as enhancer binding proteins to σ54 factor to regulate hrpL promoter • HrpL then goes on to promote other genes with hrp box • HrpV is a candidate as a negative regulator of the hrp gene cluster • Upregulated by HrpL (feedback mechanism?) ?

19. Transcriptional regulation of Pseudomonas syringae TTSS • Conserved hrp box sequence

20. Conclusion • Candidate for iGEM project? • Considerations • HrpS could function as weak activator on its own (2.5% activity) • Extend usage of pHrpL to HrpL and other effector proteins?? • HrpV needs a new promoter motif (regulated by HrpL) • Noise reduction • Requirement to strip gene cluster into individual components (other regulators involved) • Protocol for optimal media conditions • Lab techniques • RT-PCR • Microarray and RT-PCR analysis done – what other data is required (particularly with negative regulation), and how much of the project can we call our own?