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Abstract：
In yeast and higher eukaryotes, transcription factor TFIIIB is required for accurate initiation of transcription by RNA Polymerase III (Pol III), which synthesizes transfer RNAs (tRNAs), 5S ribosomal RNA (rRNA), and other essential RNA molecules. TFIIIB is composed of three subunits: B double prime 1 (Bdp1), TATA-binding protein (TBP), and TFIIB-related factor 1 (Brf1). Here, we report the molecular characterization of Brf1 in Leishmania major (LmBrf1), a parasitic protozoan that shows distinctive transcription characteristics, including the apparent absence of Pol III general transcription factors TFIIIA and TFIIIC. Although single-knockout parasites of LmBrf1 were obtained, attempts to generate LmBrf1-null mutants were unsuccessful, which suggests that LmBrf1 is essential in promastigotes of L. major. Notably, Northern blot analyses showed that the half-lives of the messenger RNAs (mRNAs) from LmBrf1 and other components of the Pol III transcription machinery (Bdp1 and Pol III subunit RPC1) are very similar (~40 min). Stabilization of these transcripts was observed in stationary-phase parasites. Chromatin immunoprecipitation (ChIP) experiments showed that LmBrf1 binds to tRNA, small nuclear RNA (snRNA), and 5S rRNA genes. Unexpectedly, the results also indicated that LmBrf1 associates to the promoter region of the 18S rRNA genes and to three Pol II-dependent regions here analyzed. Tandem affinity purification and mass spectrometry analyses allowed the identification of a putative TFIIIC subunit. Moreover, several proteins involved in transcription by all three RNA polymerases co-purified with the tagged version of LmBrf1.
摘要：
在酵母和高等真核生物中，转录因子TFIIIB是通过RNA聚合酶III（PolIII）准确启动转录所必需的，合成转移RNA（tRNA），5S核糖体RNA(rRNA),和其他必需的RNA分子。TFIIIB由三个亚基组成：B双素数1（Bdp1），TATA结合蛋白(TBP),和TFIIB相关因子1(Brf1)。这里,我们报道了主要利什曼原虫（LMBrf1）中Brf1的分子特征，一种显示独特转录特征的寄生原生动物，包括PolIII一般转录因子TFIIIA和TFIIIC的明显缺失。尽管获得了LmBrf1的单敲除寄生虫，生成LmBrf1-null突变体的尝试未成功，这表明LmBrf1在L.major的前鞭毛中必不可少。值得注意的是,Northern印迹分析表明，来自LmBrf1的信使RNA（mRNA）和PolIII转录机制的其他成分（Bdp1和PolIII亚基RPC1）的半衰期非常相似（约40分钟）。在固定相寄生虫中观察到这些转录物的稳定性。染色质免疫沉淀（ChIP）实验表明LmBrf1与tRNA结合，小核RNA（snRNA），和5SrRNA基因。出乎意料的是,结果还表明，LmBrf1与18SrRNA基因的启动子区以及本文分析的三个PolII依赖性区域相关。串联亲和纯化和质谱分析允许鉴定推定的TFIIIC亚基。此外，与标记版本的LmBrf1共纯化的所有三种RNA聚合酶参与转录的几种蛋白质。
