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及其调节剂的生化特性如何影响ParM细丝找到货物分子并主动将其推向杆状细胞两极的能力。对于这些研究，我们将结合联合收割机三种方法：（i）在体外的生化和生物物理测定ParM及其调节器的ParR/parC复合物，（ii）重建运动试验，和（iii）在体内研究的细丝组装和货物的运动。 parM基因是在许多低拷贝质粒（如R1和R100耐药质粒）上发现的分配位点（par操纵子）的一部分。为了确保质粒的拷贝在细胞分裂期间由每个子代遗传，par操纵子从三个组分构建简单的双极纺锤体：（1）称为parC的一段着丝粒DNA（Dam等人，1994）;（2）结合parC基因座的阻遏蛋白ParR;和（3）肌动蛋白样蛋白ParM（货车den Ent等，2002年）。ParR/parC复合物被认为利用ParM聚合的力来推动质粒通过细胞质（Moller-Jensen等人，2002和Moller-Jensen等人，2003年）。我们先前表征了ParM细丝的组装动力学（Garner等人，2004），并且最近，使用纯化的组分在体外重建基于ParM的DNA分离（初步结果）。据我们所知，这是第一个这样的重组任何生物系统分离DNA，它提供了一个独特的机会，了解原核生物如何使用细胞骨架系统来组织他们的细胞内空间。对于本研究，我们提出以下具体目标： 1.确定ParM组装动力学的结构和生化基础。 2.确定ParR/parC复合物促进ParM聚合的分子机制。 3.确定干扰ParM和ParR/parC复合物的生化特性对体内和体外质粒分离的准确性和效率的影响。

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.