Signaling for gene expression

1
Signaling for gene expression

• DNA Binding of PhoB and its interaction with RNA
  polymerase
• Kozo Makino, Misuko Amemura, Takeshi Kwamoto,
  Sigenobu Kimura, Hideo Shinagawa, Atsuo Nakata,
  and Masashi Suzuki

2
Phosphate-specific transportation ( Pst)system

• PhoB signal transduction response regulator in
  response to phosphate concentration in the medium

• Pi or organic phosphates passively pass through
  outer membrane pores, like PhoE porin
• Pi is released from organice phosphate compound
  in the periplasm by AP
• Pi is concentrated in the periplasm by binding to
  PBP
• Phosphate is transferred into the cytoplasm
  through the interaction with PstC, PstA and PstB.
  Energy required for this process is provided by
  ATP hydrolysis catalyzed by PstB.

Nakata, A. et al., In Phosphate Metabolism and
Cellular Regulation in Microorganisms. p150-155
3
Phosphate-specific transportation ( Pst)system

• e. Under phosphate limitation, PhoR
• is autophosphorylated and
• transfers a phosphoryl group to
• PhoB.
• When phosphate is in excess, Pst system is
  thought to form a repression complex with PhoR
  and prevents the activation of PhoB. PhoR can
  also act as a phosphatase on phospho-PhoB.
• Precise mechanism of PhoU is unknown. It is very
  likely to act by binding to PhoR, PhoB or a
  PhoR/PhoB complex as a chaperone to promote
  dephosphorylation of phospho-PhoB or by
  inhibiting formation of PhoR/PhoB transitory
  complex.

Martin G. Lamarche, et al . The phosphate regulon
and bacterial virulence a regulatory network
connecting phosphate homostasis and pathogenesis.
FEMS Microbiology Review(2008). p1-13
4
PhoB activates pho regulon expression

• PhoB activate the expression of phoA, phoE, and
  pstS activate the transport and
  assimiliation of phosphate

Nakata, A. et al., In Phosphate Metabolism and
Cellular Regulation in Microorganisms. p150-155
5
DNA Binding of PhoB and its interaction with
RNA polymerase Kozo Makino, Misuko Amemura,
Takeshi Kwamoto, Sigenobu Kimura, Hideo
Shinagawa, Atsuo Nakata, and Masashi Suzuki
6
Identification of DBD in PhoB
Figure 1
7
N terminus deletion of PhoB
Figure 2
8
Dnase I footprinting and methylation protection
Figure 3
9
Protection of the pstS prmoter by C107 from
hydroxyl radical cleavage
Figure 4
10
Effects of replacement of nucleotide bases on the
promoter activity
Figure 5
11
A possible structure of PhoB-DNA-RNA polymerase
complex
Figure 5
12
Circular Permutation analysis
Figure 6
13
Dnase I footprinting on the pstS promoter of the
four C107 mutants
Figure 7
14
Summary

• PhoB C-terminal 91 aa DBD domain
• 4 aa between Helix 2 3 important for the
  association with s70
• 4 PhoB-binding sites in the pho box in pstS
  promoter
• Upon PhoB-binding enhanced bending in pstS
  promoter

15
Further Progress
Alexandre G. B. et al. (2002), Tandem DNA
Recognition by PhoB, a Two-Component Signal
Transduction Transcriptional Activator ,
Structure, 10(5), 701-713
16
Further Progress
Alexandre G. B. et al. (2002), Tandem DNA
Recognition by PhoB, a Two-Component Signal
Transduction Transcriptional Activator ,
Structure, 10(5), 701-713