Hrp regulatory proteins, HrpR, HrpS, HrpV of Pseudomonas syringae

Closed Complex Open Complex Closed Complex Open Complex 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 Enhancer binding protein AAA+ Activator  DNA Looping Event Open Complex IHF Enhancer DNA ~ -150 Core RNAP +ATP ATP Hydrolysis activation of E  54 -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 AGAFTGAWalker BR fingerSensor II C1C7C6C5C4C3C2 HTH NH 3 COOH nucleotide binding   binding nucleotide hydrolysis intersubunit catalysis nucleotide dependent oligomerisation AAA + domain DNA binding domain C1-C7 – seven conserved sequences of  54 activators

P RS  70 PLPL other genes regulated by HrpR and HrpS LL hrpRhrpS R  54 R 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 lacZ  S R R S V - -  54 dependent - requires HrpR and HrpS proteins for activity - negatively regulated by HrpV in the presence of HrpR and HrpS proteins

activation and negative regulation of hrpL promoter MC4100  (hrpL-lacZ) 0.5mM IPTG 0.4% 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 AGAFTGAWalker BR fingerSensor II C1C7C6C5C4C3C2 HTH NH 3 COOH nucleotide binding   binding nucleotide hydrolysis intersubunit catalysis nucleotide dependent oligomerisation AAA + domain DNA binding domain C1-C7 – seven conserved sequences of  54 activators

MC4100  (hrpL-lacZ) temperature dependent activity of hrpL-lacZ fusion T ( o C) 0.5mM IPTG  54 mutant P.s. 28 o C

R lacZ  54 ? lacZ  54 S ? R R S R S S GERGTG K EL GETGTG K DT GAF T GV GAY T GA GAF T GA DE LAT DE IDS F R ADLLD R L F R RDLFF R L F R RDLYF R L WPGNI R ELKNVVE WPGNI R ELKSAAK WPGNI R ELKAAAK PspF HrpR HrpS LP RR TLYH R MKEL 228  HTH Walker AGAFTGAWalker BR fingerSensor II AAA + domain DNA binding domain C1C7C6C5C4C3C2 HTH NH3 COOH nucleotide binding   binding nucleotide hydrolysis intersubunit catalysis nucleotide dependent oligomerisation 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

Walker AGAFTGAWalker BR fingerSensor II AAA + domainDNA binding domain C1C7C6C5C4C3C2 HTH NH3 COOH GETGTGKDT GAFTGV GAYTGA DEIDS FRRDLFFRL FRRDLYFRL WPGNIRELKSAAK WPGNIRELKAAAK HrpR HrpS LPRRTLYHRMKEL  HTH red letters - mutations which abolish hrpL transcription blue letters – mutations which diminish hrpL transcription amino acids are substituted with alanine nucleotide binding   binding nucleotide hydrolysis intersubunit catalysis nucleotide dependent oligomerisation hrpL-lacZ activity in the presence of WT HrpS % + 16% % + 6% + 34% hrpL-lacZ activity in the presence of WT HrpR % functional determinants of HrpR and HrpS important for in vivo activation of the hrpL promoter in E. coli

lacZ MU IHF hrpL-lacZ transcription fusion lacZ  S R R S in vivo hrpL promoter analysis UAS

 -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 (OD 600 of ) -cool the culture(s) on ice for 20 minutes -record cell density at OD 600 -add aliquots of the culture(s) to the assay medium (Z buffer) -add 30  l of 0.1%SDS and 60  l of chloroform and vortex for 10 seconds -place the samples in a water bath at 28 O 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 Na 2 CO 3 -record the OD420 and OD550 for each sample OD 420 – (1.75 x OD 550 ) MU= 1000 x t x v x OD 600

Closed Complex Enhancer binding protein AAA+ Activator  DNA Looping Event Open Complex IHF Enhancer 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 lacZ  54 R S R S lacZ  54 R S R lacZ  54 RS 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 AGAFTGAWalker BR fingerSensor II C1C7C6C5C4C3C2 HTH NH3 COOH nucleotide binding   binding nucleotide hydrolysis intersubunit catalysis nucleotide dependent oligomerisation AAA + domainDNA binding domain GERGTGKEL GETGTGKDT GAFTGV GAYTGA GAFTGADELAT DEIDS FRADLLDRL FRRDLFFRL FRRDLYFRL WPGNIRELKNVVE WPGNIRELKSAAK WPGNIRELKAAAK PspF HrpR HrpS