Liu Wenzhe, Das Anish, Morales Rachel, Banday Mahrukh, Aris Virginie, Lukac David M, Soteropoulos Patricia, Wah David A, Palenchar Jennifer, Bellofatto Vivian
Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry-New Jersey Medical School, Newark, NJ 07103, USA.
Mol Biochem Parasitol. 2012 Dec;186(2):139-42. doi: 10.1016/j.molbiopara.2012.09.003. Epub 2012 Sep 19.
RNA polymerase II (RNAP-II) synthesizes the m(7)G-capped Spliced Leader (SL) RNA and most protein-coding mRNAs in trypanosomes. RNAP-II recruitment to DNA usually requires a set of transcription factors that make sequence-specific contacts near transcriptional start sites within chromosomes. In trypanosomes, the transcription factor TFIIB is necessary for RNAP-II-dependent SL RNA transcription. However, the trypanosomal TFIIB (tTFIIB) lacks the highly basic DNA binding region normally found in the C-terminal region of TFIIB proteins. To assess the precise pattern of tTFIIB binding within the SL RNA gene locus, as well as within several other loci, we performed chromatin immunoprecipitation/microarray analysis using a tiled gene array with a probe spacing of 10 nucleotides. We found that tTFIIB binds non-randomly within the SL RNA gene locus mainly within a 220-nt long region that straddles the transcription start site. tTFIIB does not bind within the small subunit (SSU) rRNA locus, indicating that trypanosomal TFIIB is not a component of an RNAP-I transcriptional complex. Interestingly, discrete binding sites were observed within the putative promoter regions of two loci on different chromosomes. These data suggest that although trypanosomal TFIIB lacks a highly basic DNA binding region, it nevertheless localizes to discrete regions of chromatin that include the SL RNA gene promoter.
RNA聚合酶II（RNAP-II）在锥虫中合成m(7)G帽化的剪接前导序列（SL）RNA和大多数蛋白质编码mRNA。RNAP-II募集到DNA通常需要一组转录因子，这些转录因子在染色体内转录起始位点附近进行序列特异性结合。在锥虫中，转录因子TFIIB对于依赖RNAP-II的SL RNA转录是必需的。然而，锥虫TFIIB（tTFIIB）缺乏通常在TFIIB蛋白C端区域发现的高度碱性的DNA结合区域。为了评估tTFIIB在SL RNA基因座以及其他几个基因座内的精确结合模式，我们使用探针间距为10个核苷酸的平铺基因阵列进行了染色质免疫沉淀/微阵列分析。我们发现tTFIIB在SL RNA基因座内非随机结合，主要在跨越转录起始位点的220个核苷酸长的区域内。tTFIIB不在小亚基（SSU）rRNA基因座内结合，这表明锥虫TFIIB不是RNAP-I转录复合物的组成部分。有趣的是，在不同染色体上两个基因座的推定启动子区域内观察到离散的结合位点。这些数据表明，尽管锥虫TFIIB缺乏高度碱性的DNA结合区域，但它仍然定位于包括SL RNA基因启动子的染色质离散区域。
