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Hrp regulatory proteins, HrpR, HrpS, HrpV of Pseudomonas syringae
Closed Complex
-35 -10
Open Complex
-35 -10
Closed Complex
-24 -12 +1
Open Complex
-24 -12 +1
A)70 Dependent Transcription Initiation
B)54 Dependent Transcription Initiation
Activator driven nucleotide
hydrolysis to allow RNAP
isomerisation
Spontaneous
Activator required for promoter
engagement
Ph.D., 2004, P.C. Burrows
Closed Complex
-24 -12 +1
Enhancer binding protein
AAA+ Activator
54
-24 -12 +1 -24 -12 +1
DNA Looping Event
Open Complex
IHFEnhancer
DNA ~ -150
Core RNAP
+ATP
ATP Hydrolysis
activation of E54-dependent transcription
M. Buck et al., 2006, Biochem. Soc. Transactions
C1
Central AAA+ domainRegulatory domain
C7C6C5C4C3C2
DNA binding domain
HTH
PspF
HrpS
HrpR
NH3 COOH
domain organisation of AAA+ 54 activators
domain organisation of AAA+ 54 activators lacking N-terminal domain
Walker A GAFTGA Walker B R finger Sensor II
C1 C7C6C5C4C3C2
HTH
NH3 COOH
nucleotidebinding
binding nucleotidehydrolysis
intersubunitcatalysis
nucleotide dependentoligomerisation
AAA+ domainDNA binding
domain
C1-C7 – seven conserved sequences of 54 activators
PRS
70
PL
other genesregulated byHrpR and HrpS
L
hrpR hrpS
R
54R S
The Pseudomonas syringae Hrp regulatory system
hrp regulon
S_
V
hrpL
Type III Protein Export Complex
chromosomal single copy reporter system in E. coli
hrpL-lacZ chromosomal fusion
lacZ54 +1
SR
R S
V-
- 54 dependent- requires HrpR and HrpS proteins for activity- negatively regulated by HrpV in the presence of HrpR and HrpS proteins
0
50
100
150
200
250
300
350
400b
-Gal
acto
sid
ase
acti
vity
(MU
)
activation and negative regulation of hrpL promoter
MC4100 (hrpL-lacZ)
0.5mM IPTG0.4% ara
C1
Central AAA+ domainRegulatory domain
C7C6C5C4C3C2
DNA binding domain
HTH
PspF
HrpS
HrpR
NH3 COOH
domain organisation of AAA+ 54 activators
domain organisation of AAA+ 54 activators lacking N-terminal domain
Walker A GAFTGA Walker B R finger Sensor II
C1 C7C6C5C4C3C2
HTH
NH3 COOH
nucleotidebinding
binding nucleotidehydrolysis
intersubunitcatalysis
nucleotide dependentoligomerisation
AAA+ domainDNA binding
domain
C1-C7 – seven conserved sequences of 54 activators
0
50
100
150
200
250
300
350
400
21-25 30 37
b -G
alac
tosi
das
e ac
tivi
ty (
MU
)
pAPT110hrpRS
pAPT110
MC4100 (hrpL-lacZ)
temperature dependent activity of hrpL-lacZ fusion
T (oC)
0.5mM IPTG
54 mutant
P.s. 28oC
R
lacZ54
?
lacZ54
S
?
R
R SR
S
S
GERGTGKEL
GETGTGKDT
GETGTGKDT
GAFTGV
GAYTGA
GAFTGA DELAT
DEIDS
DEIDS
FRADLLDRL
FRRDLFFRL
FRRDLYFRL
WPGNIRELKNVVE
WPGNIRELKSAAK
WPGNIRELKAAAK
PspF
HrpR
HrpS
45
22616816243 86
88
107 108
109 110 164 170 228
8642 107 108 162 168 227
LPRRTLYHRMKEL
228 228 228
HTH
Walker A GAFTGA Walker B R finger Sensor II
AAA+ domain DNA binding domain
C1 C7C6C5C4C3C2
HTH
NH3 COOH
nucleotidebinding
binding nucleotidehydrolysis
intersubunitcatalysis
nucleotide dependentoligomerisation
do both, HrpR and HrpS have to be fully functional for co-regulationwhat are the effects of changing amino acids in HrpR and HrpS
Walker A GAFTGA Walker B R finger Sensor II
AAA+ domain DNA binding domain
C1 C7C6C5C4C3C2
HTH
NH3 COOH
GETGTGKDT
GETGTGKDT
GAFTGV
GAYTGA
DEIDS
DEIDS
FRRDLFFRL
FRRDLYFRL
WPGNIRELKSAAK
WPGNIRELKAAAK
HrpR
HrpS
LPRRTLYHRMKEL
HTH
red letters - mutations which abolish hrpL transcriptionblue letters – mutations which diminish hrpL transcription
amino acids are substituted with alanine
nucleotidebinding
binding nucleotidehydrolysis
intersubunitcatalysis
nucleotide dependentoligomerisation
hrpL-lacZ activity in the presence of WT HrpS - - - ---
-- - +8%
+16%
++14%
+6%
+34%
hrpL-lacZ activity in the presence of WT HrpR
45
22616816243 86
88
107 108
109 110 164 170 228
+6%
functional determinants of HrpR and HrpS important for in vivo activation of the hrpL promoter in E. coli
-600 21458
lacZ
-100
-200
-300
MU
-400
+1
IHF -24-12
20930
21030
19200
300
10
hrpL-lacZ transcription fusion
lacZ54
+1
SR
R S
-24 -12
in vivo hrpL promoter analysis
UAS
b-Galactosidase assay
-prepare over night (O/N) culture(s)-add 0.2ml of the O/N culture(s) to the 5ml fresh media-grow cultures to mid-log phase (OD600 of 0.28-0.70)-cool the culture(s) on ice for 20 minutes-record cell density at OD600
-add aliquots of the culture(s) to the assay medium (Z buffer)-add 30l of 0.1%SDS and 60l of chloroform and vortex for 10 seconds-place the samples in a water bath at 28O for 5 minutes-start reaction by adding 0.2 ml of ONPG (4mg/ml) to each tube, shake for a few seconds-record the time of the reaction with a stop watch-stop reaction by adding 0.5ml of a 1M Na2CO3
-record the OD420 and OD550 for each sample
OD420 – (1.75 x OD550) MU= 1000 x t x v x OD600
Closed Complex
-24 -12 +1
Enhancer binding protein
AAA+ Activator
54
-24 -12 +1 -24 -12 +1
DNA Looping Event
Open Complex
IHFEnhancer
DNA ~ -150
Core RNAP
+ATP
ATP Hydrolysis
hrpL-lacZ chromosomal fusion
do both, HrpR and HrpS have to be fully functional for co-regulation
what are the effects of changing amino acids in HrpR and HrpS
R S
lacZ54R S
R S
lacZ54R S
R
lacZ54R S
S
? ?
+
C1
Central AAA+ domainRegulatory domain
C7C6C5C4C3C2
DNA binding domain
HTH
PspF
HrpS
HrpR
NH3 COOH
C1-C7 – seven conserved sequences of 54 activators
Domain organisation of AAA+ 54 activators
Domain organisation of AAA+ 54 activators lacking N-terminal domain
Walker A GAFTGA Walker B R finger Sensor II
C1 C7C6C5C4C3C2
HTH
NH3 COOH
nucleotidebinding
binding nucleotidehydrolysis
intersubunitcatalysis
nucleotide dependentoligomerisation
AAA+ domain DNA binding domain
GERGTGKEL
GETGTGKDT
GETGTGKDT
GAFTGV
GAYTGA
GAFTGA DELAT
DEIDS
DEIDS
FRADLLDRL
FRRDLFFRL
FRRDLYFRL
WPGNIRELKNVVE
WPGNIRELKSAAK
WPGNIRELKAAAK
PspF
HrpR
HrpS
45
22616816243 86
88
107 108
109 110 164 170 228
8642 107 108 162 168 227
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