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Abstract：
RNA polymerase III (RNAP III) synthetizes small essential non-coding RNA molecules such as tRNAs and 5S rRNA. In yeast and vertebrates, RNAP III needs general transcription factors TFIIIA, TFIIIB, and TFIIIC to initiate transcription. TFIIIC, composed of six subunits, binds to internal promoter elements in RNAP III-dependent genes. Limited information is available about RNAP III transcription in the trypanosomatid protozoa Trypanosoma brucei and Leishmania major, which diverged early from the eukaryotic lineage. Analyses of the first published draft of the trypanosomatid genome sequences failed to recognize orthologs of any of the TFIIIC subunits, suggesting that this transcription factor is absent in these parasites. However, a putative TFIIIC subunit was recently annotated in the databases. Here we characterize this subunit in T. brucei and L. major and demonstrate that it corresponds to Tau95. In silico analyses showed that both proteins possess the typical Tau95 sequences: the DNA binding region and the dimerization domain. As anticipated for a transcription factor, Tau95 localized to the nucleus in insect forms of both parasites. Chromatin immunoprecipitation (ChIP) assays demonstrated that Tau95 binds to tRNA and U2 snRNA genes in T. brucei. Remarkably, by performing tandem affinity purifications we identified orthologs of TFIIIC subunits Tau55, Tau131, and Tau138 in T. brucei and L. major. Thus, contrary to what was assumed, trypanosomatid parasites do possess a TFIIIC complex. Other putative interacting partners of Tau95 were identified in T. brucei and L. major. KEY POINTS: • A four-subunit TFIIIC complex is present in T. brucei and L. major • TbTau95 associates with tRNA and U2 snRNA genes • Putative interacting partners of Tau95 might include some RNAP II regulators.
摘要：
RNA聚合酶III（RNAPIII）合成小的必需非编码RNA分子，例如tRNA和5SrRNA。在酵母和脊椎动物中，RNAPIII需要一般转录因子TFIIIA，TFIIIB,和TFIIC启动转录。TFIIC,由六个亚基组成，与RNAPIII依赖性基因中的内部启动子元件结合。关于锥虫原生动物锥虫和主要利什曼原虫RNAPIII转录的信息有限，早期与真核细胞谱系不同。对首次发布的锥虫基因组序列草案的分析未能识别任何TFIIIC亚基的直向同源物，表明这种转录因子在这些寄生虫中不存在。然而,最近在数据库中注释了一个假定的TFIIIC亚基。在这里，我们描述了该亚基在T.brucei和L.major中的特征，并证明它对应于Tau95。计算机模拟分析表明，两种蛋白质都具有典型的Tau95序列：DNA结合区和二聚化结构域。正如预期的转录因子，Tau95以两种寄生虫的昆虫形式定位于细胞核。染色质免疫沉淀(ChIP)试验证明Tau95与布鲁氏菌中的tRNA和U2snRNA基因结合。值得注意的是,通过进行串联亲和纯化，我们在布鲁氏菌和主要品系中鉴定了TFIIIC亚基Tau55，Tau131和Tau138的直向同源物。因此，与假设相反，锥虫寄生虫确实具有TFIIIC复合物。Tau95的其他推定的相互作用伴侣在布鲁氏菌和L.major中鉴定出。关键点：•四亚基TFIIIC复合物存在于T.brucei和L.major•TbTau95与tRNA和U2snRNA基因相关•Tau95的推定相互作用伴侣可能包括一些RNAPII调节因子。
