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