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

2. Presentation Outline RNApol holoenzyme General properties of sigma factors The alternative σ 54 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 σ 54 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 σ 70 subunit – the generic sigma subunit

7. General Properties of σ factor

8. Bacteriophage-encoded σ factor also used to take over cellular transcriptional machinery

9. Presentation Outline RNApol holoenzyme General properties of sigma factors The alternative σ 54 factor Introduction to type III secretion system Transcriptional regulation of Pseudomonas syringae TTSS Conclusion

10. The alternative σ 54 factor Most alternative sigmas are related in sequence and structure to σ 70. 2nd distinct type of σ called the σ 54 family Differences between the σ families  σ 54 family shares no sequence homology with the σ 70 family  Whereas σ 70 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 σ 54 factor 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 Activity of the alternative σ 54 factor has been studied most intensively at the promoter for the glnA gene (encodes glutamine synthetase)

12. Presentation Outline RNApol holoenzyme General properties of sigma factors The alternative σ 54 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 σ 54 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 HrpRHrpSHrpVpHrpL 0000 1000 0100 1101 0010 1010 0110 1110

18. Transcriptional regulation of Pseudomonas syringae TTSS HrpR & HrpS forms heteromeric complex that functions as enhancer binding proteins to σ 54 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?