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CHARACTERIZING THE C-TERMINAL REGION OF PARA TO DETERMINE ITS ROLE IN REPRESSOR ACTIVITY
By: Nova Syed
Supervisor: Dr. Barbara Funnell
INTRODUCTION
INTRODUCTION
Bouet and Funnell, 1999
PAR+/REP- MUTANTS
SEPARATING THE H323Y+E332V DOUBLE MUTANT
FLOW CHART OF OVERALL CLONING STEPS
THE REPRESSION ASSAY
+β-galactosidase
ortho-Nitrophenyl-β-galactoside galactose ortho-nitrophenol
C-TERMIINAL MUTANTS DO NOT HAVE REPRESSOR ACTIVITY
0
2
4
6
8
10
12
B-G
alac
tosi
dase
A
ctiv
ity (
Mill
er U
nits
)
Grow culture overnight in
LB+Ampicillin
Plate on M9 Glu Xgal
par+
galactose 5-bromo-4-chloro- 5,5'-dibromo-4,4'-dichloro-indigo
3-hydroxyindole
THE PARTITION ASSAY
par¯
H323Y AND E332V HAVE DEFECTIVE PARTITION ACTIVITY
Partition Activity
Repressor Actvity
H323Y - -
E332V + -
E332A ++ -
H323Y+E332V ++ -
Wildtype ++ +
pBR322 (no ParA, no
ParB)
- -
- Blue
+ Light Blue
++ White
OVERVIEW OF PROTEIN PURIFICATION
Harvest Cells
Lyse By Sonication
DE-52 Column
Ni+2-Sepharose Column
Wash with low [Imidazole] Wash Buffer
Elute with high [Imidazole] Elution Buffer
Check [Protein] by Bradford Assays
SDS-PAGE OF PROTEIN PURIFICATION STEPS
t=0 t=120min FrI FrII-1 FT WI 100-3 200-1 200-2 200-3 200-4 175kDa
80
58
46
30
25
17
7
MAP OF TRYPTIC FRAGMENTS OF PARA
E332A BINDS ATP AND ADP
E332A Wildtype ParA
ELECTROPHORETIC MOBILITY SHIFT ASSAY
E332A HAS A DAMAGED SITE-SPECIFIC DNA BINDING ACTIVITY
Wildtype ParA E332A 2log DNA Ladder
parOP
No Protein
300bp
200
100
MAIN CONCLUSIONS
E332V and H323Y may be compensating mutations in the partition form of ParA
E332A mutant binds nucleotide
E332A is defective in specific binding of parOP
H323Y, E332V and E332A all have defective repressor activity
FUTURE DIRECTIONS
Further investigate the role of this C-terminal region in binding parOP
DNAseI footprinting would not only confirm gel-shift results, but also elucidate whether cooperative binding by ParA is affected.
Native gels and FRET to determine whether dimerization of ParA is compromised
The interaction between E332V and H323Y which allows only the double mutant to maintain its partition activity needs to be further examined. This would require designing a more sophisticated assay to test partition activity.
REFERENCES
Bouet,Jean-Yves and Funnell, Barbara E. P1 ParA interacts with the P1 partition complex at parS and an ATP–ADP switch controls ParA activities. 1999. The EMBO Journal Vol.18 No.5 pp.1415–1424
Davey 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 Drive Partition. Current Topics in Developmental Biology Vol. 56(pp. 145-174.). Elselvier Inc.
ACKNOWLEDGEMENTS
Dr. Barbara FunnellLori Ing
James Havey
