ELUCIDATING THE GLOBAL ARCHITECTURE OF THE BACTERIAL SEGROSOME

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 结核病，特别是耐多药的结核病，与艾滋病结合在一起，是一个严重的健康威胁。本提案的目的是了解结核分枝杆菌染色体段的PARB-DNA组装(ParABS)的三维组织。细菌中的质粒和染色体分离所需要的是底物。它由2个蛋白质组成，命名为PARB和PARB，以及一组位于复制起点附近的14-16个残基回文DNA序列PARS。 PARB在部分近端的染色体DNA上聚合，形成一个大的核蛋白组合或分配复合体(覆盖1千碱基的DNA)，在几种细菌中招募许多蛋白质，包括Para(一种驱动分离的ATPase)、SMC(染色体凝聚蛋白)、MipZ(细胞分裂位点选择器)和POPZ(细胞-极组织因子)。人们对这些细胞超结构的三维组织知之甚少，这一点在本提案中得到了解决。 由于多结构域和多聚体的性质，PARB及其络合物的结构表征是具有挑战性的。一个全长的染色体PARB以前从未结晶过。将使用溶液散射来分析PARB分区复合体的结构组织，这是了解它如何刺激对位活性并在功能上与凝聚素相互作用，导致染色体分离和凝聚的垫脚石。 Bio-SAXS站获得的PARB-DNA复合体的蔗糖对比度变化的溶液散射数据，以及其他实验数据(如Forster共振能量转移、X射线足迹和质谱仪)，将有助于建立低分辨率的藻体组织模型。对比度变化将用于突出溶液散射派生的分子包络内的两组分系统中的每一组分。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Tuberculosis, especially in its multi-drug resistant form and in combination with AIDS, is a serious health threat. The aim of this proposal is to understand three-dimensional organization of the ParB-DNA assembly of the chromosomal segrosome (ParABS) in Mycobacterium tuberculosis. The segrosome is required for plasmid and chromosome segregation in bacteria. It consists of 2 proteins, named ParA and ParB and a set of 14-16 residue palindromic DNA sequences, parS, which are found near the replication origin. ParB polymerize on the parS-proximal chromosomal DNA to form a large nucleoprotein assembly or the partition complex (covering > 1 kilobases of DNA) that recruit a number of proteins in several bacteria, including ParA (an ATPase that drives segregation), SMC (chromosome condensation protein), MipZ (cell division site selector) and PopZ (cell-pole organizing factor). Little is known about the three-dimensional organizations of these cellular superstructures, which is addressed in this proposal. Due to multi-domain and multimeric nature, structural characterization of ParB and its complexes is challenging. A full-length chromosomal ParB has never been crystallized before. Architectural organization of the ParB partition complex will be analyzed using solution scattering, which is a stepping-stone to understand how it stimulates ParA activity and functionally interacts with condensins, leading to chromosome segregation and condensation. Solution scattering data with sucrose contrast variation on the ParB-DNA complex obtained from Bio-SAXS station, together with other experimental data (such as Forster resonance energy transfer, X-ray footprinting with mass spectrometry), will assist in building low-resolution model of segrosome organization. Contrast variations will serve to highlight each component in a two-component system within the solution scattering-derived molecular envelope.