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

2. 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

3. Closed Complex -24 -12 +1 Enhancer binding protein AAA+ Activator  54 -24 -12 +1 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

4. 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

5. 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

6. chromosomal single copy reporter system in E. coli hrpL-lacZ chromosomal fusion lacZ  54 +1 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

7. activation and negative regulation of hrpL promoter MC4100  (hrpL-lacZ) 0.5mM IPTG 0.4% ara

8. 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

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

10. 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 45 226 168162 43 86 88 107 108 109 110 164 170 228 86 42 107 108162168227 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

11. 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 - --- - - -- - + 8% + 16% + + 14% + 6% + 34% hrpL-lacZ activity in the presence of WT HrpR 45 226 168162 43 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

12. -600 21458 lacZ -100 -200 -300 MU -400 +1 IHF -24-12 20930 21030 19200 300 10 hrpL-lacZ transcription fusion lacZ  54 +1 S R R S -24 -12 in vivo hrpL promoter analysis UAS

13.  -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 0.28-0.70) -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

15. Closed Complex -24 -12 +1 Enhancer binding protein AAA+ Activator  54 -24 -12 +1 DNA Looping Event Open Complex IHF Enhancer DNA ~ -150 Core RNAP +ATP ATP Hydrolysis

16. 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 ? ? +

17. 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 45 226 168162 43 86 88 107 108 109 110 164 170 228 86 42 107 108162168227