Department of Animal Biology, School of Life Sciences, University of Hyderabad, Hyderabad, India.
Department of Animal Biology, School of Life Sciences, University of Hyderabad, Hyderabad, India.
Biochim Biophys Acta Gen Subj. 2022 Dec;1866(12):130240. doi: 10.1016/j.bbagen.2022.130240. Epub 2022 Sep 2.
The mRNA transcription is a multistep process involving distinct sets of proteins associated with RNA polymerase II (RNAPII) through various stages. Recent studies have highlighted the role of RNAPII-associated proteins in facilitating the assembly of functional complexes in a crowded nuclear milieu. RNAPII dynamics and gene expression regulation have been primarily studied in model eukaryotes like yeasts and mammals and remain largely unchartered in protozoan parasites like Toxoplasma gondii, where considerable gene expression changes accompany stage differentiations. Here we report a key modulator of RNAPII activity, TFIIS in Toxoplasma gondii (TgTFIIS).
A Pull-down assay demonstrated that TgTFIIS binds to RNAPII subunit TgRPB1. Truncation mutants of TFIIS help us define the regions critical for its binding to TgRPB1. Co-immunoprecipitation analysis confirmed the interaction between the native TgTFIIS and TgRPB1. Confocal microscopy revealed a predominantly nuclear localization. Native TgTFIIS was able to bind promoter DNA which was consistent with the CHIP results.
TgTFIIS complements initiation defects in yeast mutants, and the regions implicated in RNAPII binding appeared essential for this function. Interestingly, the C-terminal zinc finger domain necessary for its potential elongation function is dispensable for TgRPB1 binding. TgTFIIS was found to be associated with the promoter region along with its association with the ORF on an RNAPII transcribed gene.
The observations were in line with the potential role of TgTFIIS in early events of RNAPII transcription in addition to elongation.
The study elucidates the potential role of RNAPII-associated proteins in multiple steps of transcription.
mRNA 转录是一个多步骤的过程，涉及与 RNA 聚合酶 II（RNAPII）通过不同阶段相关的不同蛋白质组。最近的研究强调了 RNAPII 相关蛋白在促进功能复合物在拥挤的核环境中组装的作用。RNAPII 的动力学和基因表达调控主要在模式真核生物（如酵母和哺乳动物）中进行研究，而在原生动物寄生虫（如刚地弓形虫）中仍在很大程度上未被探索，在这些寄生虫中，伴随着阶段分化会发生相当大的基因表达变化。在这里，我们报道了刚地弓形虫中 RNAPII 活性的一个关键调节剂，即 TFIIS（TgTFIIS）。
Pull-down 实验表明，TgTFIIS 与 RNAPII 亚基 TgRPB1 结合。TFIIS 的截断突变体帮助我们确定了其与 TgRPB1 结合的关键区域。共免疫沉淀分析证实了天然 TgTFIIS 和 TgRPB1 之间的相互作用。共聚焦显微镜显示主要定位于核内。天然 TgTFIIS 能够结合启动子 DNA，这与 CHIP 结果一致。
TgTFIIS 弥补了酵母突变体中的起始缺陷，并且与 RNAPII 结合相关的区域对于该功能似乎是必需的。有趣的是，其潜在延伸功能所需的 C 端锌指结构域对于 TgRPB1 结合是可有可无的。发现 TgTFIIS 与启动子区域以及与正在转录的基因的 ORF 相关联。
这些观察结果与 TgTFIIS 在 RNAPII 转录的早期事件中除延伸之外的潜在作用一致。
该研究阐明了 RNAPII 相关蛋白在转录多个步骤中的潜在作用。
