Regulation and Function of a Bacterial Cytoskeleton

描述（由申请人提供）：本提案的目标是表征原核肌动蛋白样蛋白ParM的结构和组装动力学，并了解ParM及其调节因子的生化特性如何影响ParM细丝寻找货物分子并主动将其推向杆状细胞极点的能力。对于这些研究，我们将结合三种方法：(i) ParM及其调节因子ParR/parC复合物的体外生化和生物物理分析，（ii）重组运动性分析，以及（iii）纤维组装和货物运动的体内研究。

DESCRIPTION (provided by applicant): The goal of the present proposal is to characterize the structure and assembly dynamics of the prokaryotic actin-like protein, ParM, and to understand how the biochemical properties of ParM and its regulators affect the ability of ParM filaments to find cargo molecules and actively push them to the poles of rod-shaped cells. For these studies we will combine three approaches: (i) in vitro biochemical and biophysical assays on ParM and its regulator the ParR/parC complex, (ii) reconstituted motility assays, and (iii) in vivo studies of filament assembly and cargo movement. The parM gene is part of a partitioning locus (the par operon) found on many low-copy plasmids, such as the R1 and R100 drug-resistance plasmids. To ensure that a copy of the plasmid is inherited by each daughter during cell division, the par operon constructs a simple bipolar spindle from three components: (1) a stretch of centromeric DNA called parC (Dam et al., 1994); (2) a repressor protein, ParR, that binds the parC locus; and (3) the actin-like protein ParM (van den Ent et al., 2002). The ParR/parC complex is thought to harness the force of ParM polymerization to push plasmids through the cytoplasm (Moller-Jensen et al., 2002 and Moller-Jensen et al., 2003). We previously characterized the assembly dynamics of ParM filaments (Garner et al., 2004) and, more recently, reconstituted ParM-based DNA segregation in vitro using purified components (Preliminary Results). To our knowledge, this is the first such reconstitution of any biological system for segregating DNA and it provides a unique opportunity to understand how prokaryotes use cytoskeletal systems to organize their intracellular spaces. For the present study, we propose the following specific aims: 1. Determine the structural and biochemical bases of ParM assembly dynamics. 2. Determine the molecular mechanism by which the ParR/parC complex promotes ParM polymerization. 3. Determine the effect of perturbing the biochemical properties of ParM and the ParR/parC complex on the accuracy and efficiency of plasmid segregation in vivo and in vitro.