CNRS, CEA, IBS, University Grenoble Alpes, 38000 Grenoble, France.
CNRS, CEA, INRAE, IRIG-LPCV, University Grenoble-Alpes, 38000 Grenoble, France.
Int J Mol Sci. 2022 Aug 31;23(17):9922. doi: 10.3390/ijms23179922.
RNA polymerases (RNAPs) are found in all living organisms. In the chloroplasts, the plastid-encoded RNA polymerase (PEP) is a prokaryotic-type multimeric RNAP involved in the selective transcription of the plastid genome. One of its active states requires the assembly of nuclear-encoded PEP-Associated Proteins (PAPs) on the catalytic core, producing a complex of more than 900 kDa, regarded as essential for chloroplast biogenesis. In this study, sequence alignments of the catalytic core subunits across various chloroplasts of the green lineage and prokaryotes combined with structural data show that variations are observed at the surface of the core, whereas internal amino acids associated with the catalytic activity are conserved. A purification procedure compatible with a structural analysis was used to enrich the native PEP from chloroplasts. A mass spectrometry (MS)-based proteomic analysis revealed the core components, the PAPs and additional proteins, such as FLN2 and pTAC18. MS coupled with crosslinking (XL-MS) provided the initial structural information in the form of protein clusters, highlighting the relative position of some subunits with the surfaces of their interactions. Using negative stain electron microscopy, the PEP three-dimensional envelope was calculated. Particles classification shows that the protrusions are very well-conserved, offering a framework for the future positioning of all the PAPs. Overall, the results show that PEP-associated proteins are firmly and specifically associated with the catalytic core, giving to the plastid transcriptional complex a singular structure compared to other RNAPs.
RNA 聚合酶（RNAPs）存在于所有生物体中。在叶绿体中，质体编码的 RNA 聚合酶（PEP）是一种参与质体基因组选择性转录的原核型多聚体 RNAP。其活性状态之一需要核编码的 PEP 相关蛋白（PAPs）在催化核心上组装，形成一个超过 900 kDa 的复合物，被认为对叶绿体发生至关重要。在这项研究中，对绿色谱系和原核生物中各种叶绿体的催化核心亚基进行序列比对，并结合结构数据表明，核心表面存在变异，而与催化活性相关的内部氨基酸则保守。采用与结构分析兼容的纯化程序从叶绿体中富集天然 PEP。基于质谱（MS）的蛋白质组学分析揭示了核心成分、PAPs 和其他蛋白质，如 FLN2 和 pTAC18。MS 与交联（XL-MS）的结合提供了蛋白质簇形式的初始结构信息，突出了一些亚基与相互作用表面的相对位置。使用负染色电子显微镜计算了 PEP 的三维包络。颗粒分类表明突起非常保守，为未来所有 PAPs 的定位提供了一个框架。总的来说，结果表明 PEP 相关蛋白与催化核心牢固且特异性地结合，赋予质体转录复合物与其他 RNAP 相比独特的结构。
