Sigma Factors

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Sigma Factors Transcriptional Regulation of P.
syringae TTSS

• Alexander Wong

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Presentation Outline

• RNApol holoenzyme
• General properties of sigma factors
• The alternative s54 factor
• Introduction to type III secretion system
• Transcriptional regulation of Pseudomonas
  syringae TTSS
• Conclusion

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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 (s) factor.

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The RNApol holoenzyme

• All multi-subunit RNA polymerases have 5 core
  subunits.
• Bacterial RNApol have additional s subunit
• Has function in binding to promoter
• In bacteria, RNApol binds a promoter via s
• In eukaryotes, RNApol binds via TF complex
• Bacterial RNApol is regulated purely by s
  (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.

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Presentation Outline

• RNApol holoenzyme
• General properties of sigma factors
• The alternative s54 factor
• Introduction to type III secretion system
• Transcriptional regulation of Pseudomonas
  syringae TTSS
• Conclusion

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General Properties of s factor

• RNA polymerase holoenzyme binds directly to DNA
  via its s 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 s70 subunit the generic
  sigma subunit

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General Properties of s factor
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General Properties of s factor

• Bacteriophage-encoded s factor also used to take
  over cellular transcriptional machinery

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Presentation Outline

• RNApol holoenzyme
• General properties of sigma factors
• The alternative s54 factor
• Introduction to type III secretion system
• Transcriptional regulation of Pseudomonas
  syringae TTSS
• Conclusion

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The alternative s54 factor

• Most alternative sigmas are related in sequence
  and structure to s 70.
• 2nd distinct type of s called the s54 family
• Differences between the s families
• s 54 family shares no sequence homology with the
  s 70 family
• Whereas s 70 holoenzymes carry out this process
  of open complex formation on their own, s 54
  holoenyzmes require both an enhancer and ATP to
  perform this process.

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The alternative s54 factor

• Activity of the alternative s54 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

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Presentation Outline

• RNApol holoenzyme
• General properties of sigma factors
• The alternative s54 factor
• Introduction to type III secretion system
• Transcriptional regulation of Pseudomonas
  syringae TTSS
• Conclusion

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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

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

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Presentation Outline

• RNApol holoenzyme
• General properties of sigma factors
• The alternative s54 factor
• Introduction to type III secretion system
• Transcriptional regulation of Pseudomonas
  syringae TTSS
• Conclusion

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Transcriptional regulation of Pseudomonas
syringae TTSS
?
HrpR HrpS HrpV
pHrpL
HrpR HrpS HrpV pHrpL
0 0 0 0
1 0 0 0
0 1 0 0
1 1 0 1
0 0 1 0
1 0 1 0
0 1 1 0
1 1 1 0
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Transcriptional regulation of Pseudomonas
syringae TTSS
?

• HrpR HrpS forms heteromeric complex that
  functions as enhancer binding proteins to s54
  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?)

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Transcriptional regulation of Pseudomonas
syringae TTSS

• Conserved hrp box sequence

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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?