4th Year Thesis

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1. CHARACTERIZING THE C-TERMINALREGION OF PARA TO DETERMINE ITSROLE IN REPRESSOR ACTIVITYBy: Nova SyedSupervisor: Dr. Barbara Funnell 2. INTRODUCTION 3. INTRODUCTIONBouet and Funnell, 1999 4. PAR+/REP- MUTANTS 5. SEPARATING THE H323Y+E332V DOUBLE MUTANT 6. FLOW CHART OF OVERALL CLONING STEPS 7. THE REPRESSION ASSAY-galactosidase+ortho-Nitrophenyl--galactoside galactose ortho-nitrophenol 8. C-TERMIINAL MUTANTS DO NOT HAVE REPRESSOR ACTIVITY 2000 1800 1600 1400B-Galactosidase Activity (Miller Units) 1200 10008006004002000WT ParA+ParB H323Y+E332V H323Y +WTParB E332V +WTParB E332A +WTParB WT ParA noParA no ParB +WTParB 9. THE PARTITION ASSAYGrow culture overnight inLB+AmpicillinPlate on M9 Glu Xgalgalactose 5-bromo-4-chloro- 5,5-dibromo-4,4-dichloro-indigo 3-hydroxyindolepar+par 10. H323Y AND E332V HAVE DEFECTIVEPARTITION ACTIVITY Partition Activity Repressor Actvity H323Y ---Blue E332V +- +Light Blue++ White E332A++- H323Y+E332V++-Wildtype++ +pBR322 --(no ParA, no ParB) 11. OVERVIEW OF PROTEIN PURIFICATIONHarvest CellsLyse By SonicationDE-52 Column Ni+2-Sepharose ColumnWash with low [Imidazole] Wash BufferElute with high [Imidazole] Elution Buffer Check [Protein] by Bradford Assays 12. SDS-PAGE OF PROTEIN PURIFICATION STEPS t=0 t=120minFrI FrII-1 FT WI 100-3 200-1 200-2 200-3 200-4175kDa8058463025177 13. MAP OF TRYPTIC FRAGMENTS OF PARA 14. E332A BINDS ATP AND ADPE332AWildtype ParA 15. ELECTROPHORETIC MOBILITY SHIFT ASSAY 16. E332A HAS A DAMAGED SITE-SPECIFIC DNABINDING ACTIVITYWildtype ParA E332A 2log DNANoLadderProteinparOP 300bp200100 17. MAIN CONCLUSIONSH323Y, E332V and E332A all have defective repressor activity E332V and H323Y may be compensating mutations in the partition form of ParA E332A mutant binds nucleotideE332A is defective in specific binding of parOP 18. FUTURE DIRECTIONS Further investigate the role of this C-terminal region in binding parOP DNAseI footprinting would not only confirm gel-shift results, but alsoelucidate whether cooperative binding by ParA is affected. Native gels and FRET to determine whether dimerization of ParA iscompromised The interaction between E332V and H323Y which allows only thedouble mutant to maintain its partition activity needs to be furtherexamined. This would require designing a more sophisticated assayto test partition activity. 19. REFERENCESBouet,Jean-Yves and Funnell, Barbara E. P1 ParA interacts with the P1 partition complex at parS and an ATPADP switch controls ParA activities. 1999. The EMBO Journal Vol.18 No.5 pp.14151424Davey MJ, Funnell BE. Modulation of the P1 plasmid partition protein ParA by ATP, ADP, and P1 ParB. J Biol Chem. 1997 Jun 13;272(24):15286-92.Dunham TD, Xu W, Funnell BE, Schumacher MA. Structural basis for ADP-mediated transcriptional regulation by P1 and P7 ParA. EMBO J. 2009 Jun 17;28(12):1792-802. Epub 2009 May 21.Fung E, Bouet JY, Funnell BE. Probing the ATP-binding site of P1 ParA: partition and repression have different requirements for ATP binding and hydrolysis. EMBO J. 2001 Sep 3;20(17):4901-11.Funnell, B, & Slavcev, RA. (2004). Plasmid biology: Ch 5: Partition systems of bacterial plasmids. Washington, DC: ASM Press.Surtees, J. A., & Funnell, B. E. (2003). Plasmid and Chromosome Traffic Control: How ParA and ParB DrivePartition. Current Topics in Developmental Biology Vol. 56(pp. 145-174.). Elselvier Inc. 20. ACKNOWLEDGEMENTS Dr. Barbara Funnell Lori IngJames Havey