Transcript Sigma Factors & the Hrp

Sigma Factors &
Transcriptional Regulation of
P. syringae TTSS
Alexander Wong
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
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The RNApol holoenzyme
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Definition of holoenzyme
 Complete,
working version of an enzyme
 cf. apoenzyme - missing specific cofactors that allow
it to perform its job
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Examples of cofactors
 common
prosthetic groups (haem) or metal ions
(magnesium)
 Dissociable protein subunits – sigma (σ) factor.
The RNApol holoenzyme
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All multi-subunit RNA polymerases have 5 core subunits.
Bacterial RNApol have additional σ subunit
Has function in binding to promoter
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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.
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
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General Properties of σ factor
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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
General Properties of σ factor
General Properties of σ factor
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Bacteriophage-encoded σ factor also used to
take over cellular transcriptional machinery
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
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The alternative σ54 factor
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Most alternative sigmas are related in sequence
and structure to σ 70.
2nd distinct type of σ called the σ54 family
Differences between the σ families
 σ 54
family shares no sequence homology with the σ
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.
70
The alternative σ54 factor
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Activity of the alternative σ54 factor has been studied most intensively
at the promoter for the glnA gene (encodes glutamine synthetase)
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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
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
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Introduction to type III secretion
system (TTSS)
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System with many names – PEC, injectisome, TTSS, TTS etc.
Function to deliver bacterial proteins into target cells that then
modulate host cell functions
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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
Introduction to type III secretion
system (TTSS)
Example: S. typhimurium TTSS1
hrp pathogenicity island
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Shaded genes involved in regulatory functions
hrp box – promoter motif of HrpL
Expression of hrp genes induced by:
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Pathogenesis
Acidic minimal salts medium
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
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Transcriptional regulation of
Pseudomonas syringae TTSS
HrpR
pHrpL
HrpS
HrpV
?
HrpR
HrpS
HrpV
pHrpL
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0
1
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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
Transcriptional regulation of
Pseudomonas syringae TTSS
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HrpR & HrpS forms heteromeric complex that functions as enhancer
binding proteins to σ54 factor to regulate hrpL promoter
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HrpL then goes on to promote other genes with hrp box
HrpV is a candidate as a negative regulator of the hrp gene cluster
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Upregulated by HrpL (feedback mechanism?)
Transcriptional regulation of
Pseudomonas syringae TTSS
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Conserved hrp box sequence
Conclusion
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Candidate for iGEM project?
Considerations
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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
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Requirement to strip gene cluster into individual components (other regulators
involved)
 Protocol for optimal media conditions
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Lab techniques
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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?