SAXS OF PROHEAD RNA (PRNA) OF BACTERIOPHAGE O29 DNA PACKAGING MOTOR

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。 本项目的目的是确定噬菌体29DNA包装马达的前头RNA(PRNA)的多聚体状态。 枯草杆菌噬菌体29的PRNA是噬菌体基因组的174个碱基的转录本，是29DNA包装马达的重要组成部分。多个拷贝的PRNA与前体蛋白外壳(ProHead)的头部-尾部连接器结合。ATPase gp16随后与PRNA结合构成了19千碱基对DNA-基因产物3复合体(DNA-GP3)易位的马达。遗传实验表明，相同的PRNA分子通过伪结连接在一起，形成了六聚体结构[1，2]。然而，CryoEM-3D重建表明，当PRNA连接到探针上时，它具有5次对称性[3-5]。我们已经通过核磁共振光谱确定了PRNA假结中的分子间氢键，目前正在确定PRNA假结的三维结构，这代表了RNA世界中一个新的结构基序。我们将通过使用核磁共振测量剩余偶极耦合来获得环中单个PRNA分子的相对取向。我们需要进行SAXS实验来验证环结构中有多少PRNA分子。通过结合假结结构、单个分子的排列和环中分子的数量，我们将构建PRNA环的三维结构。

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. The objective of this project is to determine the multimeric state of the prohead RNA (pRNA) of bacteriophage ¿29 DNA packaging motor. pRNA of the Bacillus subtilis bacteriophage ¿29, a 174-base transcript of the phage genome, is an essential component of the ¿29 DNA packaging motor. Multiple copies of pRNA bind to the head-tail connector of the precursor protein shell (prohead). Subsequent binding of the ATPase gp16 to pRNA constitutes the motor that translocates the 19 kilobase pair DNA-gene product 3 complex (DNA-gp3). Genetic experiments have suggested that identical pRNA molecules are linked through pseudoknots resulting in a hexameric structure [1, 2]. However, cryoEM-3D reconstruction has shown that pRNA has 5-fold symmetry when it attached to the prohead [3-5]. We have identified intermolecular hydrogen bonds within the pseudoknot of pRNA by using NMR spectroscopy, and are currently determining the three dimensional structure of the pseudoknot of pRNA, which represents a new structural motif in the RNA world. We will obtain relative orientation of individual pRNA molecules in the ring by measuring residual dipolar couplings using NMR. We need to perform SAXS experiments to verify how many pRNA molecules are in the ring structure. By combining the pseudoknot structure, the alignment of individual molecules and the number of molecules in the ring, we will build the three dimensional structure of the pRNA ring.