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