Abstract
ParA, a P1 protein required for partition of the prophage plasmid, regulates expression of its own gene and another partition gene, parB, from a promoter upstream of parA. The ATP-dependent ParA DNA binding activity to the par promoter is thought to mediate this regulation. An alternate purification for ParA is presented. This highly purified ParA was used to examine ParA DNA binding activity using DNase I protection assays. At high concentration, ParA bound to the par promoter in the absence of ATP, demonstrating that although ATP stimulates, it is not required for DNA binding. Non-hydrolyzable ATP analogues as well as ADP stimulated binding more than ATP, suggesting that the act of hydrolysis is coupled to release of the DNA. Glycerol gradient sedimentation and chemical cross-linking experiments suggest that ParA exists in an monomer-dimer equilibrium that is shifted toward dimer formation by adding ATP or ADP. These observations lead to the proposal that the more active DNA binding form of ParA is a dimer and that the effects of ATP and ParA concentration on DNA binding are a direct result of their effects on oligomerization.
Highlights
From the Department of Molecular and Medical Genetics, University of Toronto, i’bronto, Ontario, Canada M5S 1A8
Par& a P1 protein required for partition of the pro- pression even further, ParB alone has no repressor phage plasmid, regulates expressionitsofown gene and activity [5]
DNA bindingthetopar promoter was not are fully protected from DNaseI cleavage (Figs. 2 and 3)
Summary
99% homogeneity, as judged by Coomassie Blue-stained SDS- sequence, including the 20-bp inverted repeat. DNA bindingthetopar promoter was not are fully protected from DNaseI cleavage (Figs. 2 and 3)
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