Regulation and Function of a Bacterial Cytoskeleton

描述(由申请人提供)：本提案的目的是描述原核生物肌动蛋白样蛋白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细丝发现货物分子并将其主动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复合体的体外生化和生物物理分析，(Ii)重组运动分析，以及(Iii)体内细丝组装和货物移动的研究。 Parm基因是在许多低拷贝质粒上发现的分割基因(PAR操纵子)的一部分，例如R1和R100耐药质粒。为了确保在细胞分裂过程中每个子代都继承了一份质粒拷贝，PAR操纵子由三个成分组成一个简单的两极纺锤体：(1)一段称为ParC的着丝粒DNA(Dam等人，1994)；(2)一种与ParC位点结合的阻遏蛋白PARR；以及(3)类肌动蛋白Parm(van den Ent等人，2002年)。Parr/Parc复合体被认为利用Parm聚合的力量推动质粒穿过细胞质(Moller-Jensen等人，2002和Moller-Jensen等人，2003)。我们以前表征了Parm细丝的组装动力学(Garner等人，2004年)，最近，我们使用纯化的成分在体外重新构建了基于Parm的DNA分离(初步结果)。据我们所知，这是第一次这样的生物系统分离DNA，它提供了一个独特的机会来了解原核生物如何利用细胞骨架系统来组织它们的细胞内空间。就本研究而言，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复合体促进PARM聚合的分子机制。 3.测定体内、外干扰PARM和PARR/PARC复合体的生化性质对质粒分离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.