Structural studies on the P1 plasmid partition apparatus.

P1质粒分配装置的结构研究。

• 批准号: 7190843
• 负责人: Maria Schumacher
• 金额: $ 18.18万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-28 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7190843
• 关键词: DNA binding protein; Escherichia coli; adenosinetriphosphatase; bacterial genetics; calorimetry; chimeric proteins; chromosome movement; crystallization; fluorescence polarization; functional /structural genomics; intermolecular interaction; molecular dynamics; nucleic acid structure; plasmids; protein protein interaction; transcription factor

DESCRIPTION (provided by applicant): The survival of any species demands the faithful inheritance of genetic information. Essential to this process are the directed movements and positioning of chromosomes or plasmids such that they are accurately distributed to the daughter cells at cell division. Although prokaryotes do not undergo the complex mitotic steps associated with eukaryotic cells, prokaryotic chromosomes are nevertheless dynamically arranged during the cell cycle via the action of segregation, or par systems. Coordination of this process requires precise protein-DNA and protein-protein interactions carried out by par systems. All known plasmid encoded par loci specify three components: a cis-acting centromere site (parS), and two proteins, ParB and ParA. In the E. coli P1 par system, which has served as a paradigm for understanding partition, the genes for ParA and ParB form an operon, and the approximately 74 bp partition site, parS, is located immediately downstream of parB . ParB, a 38 kDa protein with no sequence homology to any protein, is a DNA-binding protein and, along with IMF or alone, binds to a highly complex centromere-like site, parS, to form the partition complex. ParA, a 44 kDa Walker-type ATPase, utilizes the energy of ATP hydrolysis to drive plasmid separation after interacting with ParB in the partition complex. Our recent structure determinations of ParB and the minimal partition site have revealed novel DNA-binding characteristics of ParB that explain its ability to bind, spread and pair plasmids. Thus, these structures have provided unprecedented insight into the mechanism of partition complex formation. Completely unknown, however, are structural bases for plasmid separation, the step carried out by ParA. Thus, in this proposal we will build on our recent progress towards a full elucidation of P1 partition with the following Specific Aims: (1) fully elucidate the mechanism of P1 partition complex formation through structural and biochemical studies on the ParB-parS partition complex. (2) Clarify the mechanism of P1 plasmid separation via structural and biochemical studies on the key end states of P1 ParA (apoParA, ParA-ADP and the ParA(K188Q)-(AMP-PCP)-ParB(1-28) complex). These structures will provide the foundation for understanding how partition systems function to drive chromosome segregation in prokaryotes and may provide potential points of therapeutic intervention against pathogenic bacteria, which also depend on par systems for segregation and thus, survival.

描述（由申请人提供）：任何物种的生存都需要忠实的遗传信息继承。这一过程至关重要的是染色体或质粒的定向运动和定位，以便将它们准确地分布在细胞分裂的子细胞上。尽管原核生物没有经历与真核细胞相关的复杂有丝分裂步骤，但仍通过隔离或PAR系统的作用在细胞周期中动态排列原核染色体。该过程的配位需要PAR系统进行的精确蛋白-DNA和蛋白质 - 蛋白质相互作用。所有已知的质粒编码PAR基因座均指定三个组成部分：顺式作用中心位点（PARS）和两个蛋白质，PARB和PARA。在充当理解分区范式的大肠杆菌P1 PAR系统中，Para和Parb的基因形成了操纵子，大约74个BP分区位点PARS位于PARB的下游。 PARB是一种38 kDa蛋白，与任何蛋白质无序性同源性，是一种DNA结合蛋白，与IMF或单独使用，与高度复杂的中心粒状位点结合，形成分区复合物。 Para是一种44 kDa Walker型ATPase，它利用ATP水解的能量在分区复合物中与PARB相互作用后驱动质粒分离。我们最近对PARB和最小分区位点的结构确定揭示了PARB的新型DNA结合特征，这些特征解释了其结合，扩散和配对质粒的能力。因此，这些结构为分区复合物形成机理提供了前所未有的见解。然而，完全未知的是用于质粒分离的结构碱基，这是由Para进行的步骤。因此，在这项建议中，我们将基于以下特定目的全面阐明P1分区的最新进展：（1）通过对PARB-PARS分区络合物的结构和生化研究，充分阐明了P1分区复合物形成的机制。 （2）通过在P1 Para（Apopara，Para-ADP和Para（K188Q） - （AMP-PCP）-Parb（1-28）复合物的P1 para（Apopara，Para-ADP）和Para（K188Q）的关键端状态下，通过结构和生化研究阐明了P1质粒分离的机制。这些结构将为理解分区系统在原核生物中驱动染色体隔离的功能提供基础，并可能提供针对致病细菌的治疗干预的潜在点，这也取决于用于隔离的PAR系统，从而依靠生存。