The small mitochondrial genome (mtDNA) of the malaria parasite is known to transcribe its genes polycistonically, although promoter element(s) have not yet been identified. An unusually large Plasmodium falciparum candidate mitochondrial phage-like RNA polymerase (PfmtRNAP) with an extended N-terminal region is encoded by the parasite nuclear genome. Using specific antibodies against the enzyme, we established that PfmtRNAP was targeted exclusively to the mitochondrion and interacted with mtDNA. Phylogenetic analysis showed that it is part of a separate apicomplexan clade. A search for PfmtRNAP-associated transcription initiation factors using sequence homology and in silico protein–protein interaction network analysis identified PfKsgA1. PfKsgA1 is a dual cytosol- and mitochondrion-targeted protein that functions as a small subunit rRNA dimethyltransferase in ribosome biogenesis. Chromatin immunoprecipitation showed that PfKsgA1 interacts with mtDNA, and in vivo crosslinking and pull-down experiments confirmed PfmtRNAP-PfKsgA1 interaction. The ability of PfKsgA1 to serve as a transcription initiation factor was demonstrated by complementation of yeast mitochondrial transcription factor Mtf1 function in Rpo41-driven in vitro transcription. Pull-down experiments using PfKsgA1 and PfmtRNAP domains indicated that the N-terminal region of PfmtRNAP interacts primarily with the PfKsgA1 C-terminal domain with some contacts being made with the linker and N-terminal domain of PfKsgA1. In the absence of full-length recombinant PfmtRNAP, solution structures of yeast mitochondrial RNA polymerase Rpo41 complexes with Mtf1 or PfKsgA1 were determined by small-angle X-ray scattering. Protein interaction interfaces thus identified matched with those reported earlier for Rpo41-Mtf1 interaction and overlaid with the PfmtRNAP-interfacing region identified experimentally for PfKsgA1. Our results indicate that in addition to a role in mitochondrial ribosome biogenesis, PfKsgA1 has an independent function as a transcription initiation factor for PfmtRNAP.

已知疟疾寄生虫的小线粒体基因组 (mtDNA) 可多顺式转录其基因，尽管尚未确定启动子元件。异常大的恶性疟原虫候选线粒体噬菌体样 RNA 聚合酶 ( Pf mtRNAP) 具有扩展的 N 末端区域，由寄生虫核基因组编码。使用针对该酶的特异性抗体，我们确定Pf mtRNAP 专门针对线粒体并与 mtDNA 相互作用。系统发育分析表明它是一个单独的顶复门进化枝的一部分。使用序列同源性和计算机蛋白质-蛋白质相互作用网络分析寻找Pf mtRNAP 相关转录起始因子Pf KsgA1。Pf KsgA1 是一种双重靶向细胞质和线粒体的蛋白质，在核糖体生物发生中充当小亚基 rRNA 二甲基转移酶。染色质免疫沉淀显示Pf KsgA1 与 mtDNA 相互作用，体内交联和下拉实验证实了Pf mtRNAP- Pf KsgA1 相互作用。Pf KsgA1 作为转录起始因子的能力通过酵母线粒体转录因子 Mtf1 功能在 Rpo41 驱动的体外转录中的互补来证明。下拉使用实验Pf的KsgA1和Pf的mtRNAP域表示的N末端区域Pf的mtRNAP主要与交互Pf的一些触点与所述连接子和的N-末端结构域由KsgA1 C-末端结构域Pf的KsgA1。在没有全长重组Pf mtRNAP的情况下，酵母线粒体 RNA 聚合酶 Rpo41 与 Mtf1 或Pf KsgA1复合物的溶液结构通过小角度 X 射线散射确定。由此鉴定与那些匹配蛋白相互作用接口先前针对Rpo41-MTF1相互作用报道并与覆盖Pf的用于实验鉴定mtRNAP-接口区域Pf的KsgA1。我们的结果表明，除了在线粒体核糖体生物发生中的作用外，PfKsgA1 作为Pf mtRNAP的转录起始因子具有独立的功能。