Das Anish, Li Hong, Liu Tong, Bellofatto Vivian
Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, International Center for Public Health, 225 Warren Street, Newark, NJ 07103, USA.
Mol Biochem Parasitol. 2006 Dec;150(2):201-10. doi: 10.1016/j.molbiopara.2006.08.002. Epub 2006 Aug 28.
In Trypanosoma brucei, transcription by RNA polymerase II accounts for the expression of the spliced leader (SL) RNA and most protein coding mRNAs. To understand the regulation of RNA polymerase II transcription in these parasites, we have purified a transcriptionally active enzyme through affinity chromatography of its essential subunit, RPB4. The enzyme preparation is active in both promoter-independent and promoter-dependent in vitro transcription assays. Importantly, the enzyme is sensitive to alpha-amanitin inhibition, a hallmark of eukaryotic RNA polymerase II enzymes. Using mass spectrometric analysis we have identified the previously unobserved RPB12 subunit of T. brucei RNA polymerase II. TbRPB12 contains a conserved CX(2)CX(10-15)CX(2)C zinc binding motif that is characteristic of other eukaryotic RPB12 polypeptides. We also identified seven proteins that associate with T. brucei RNA polymerase II. While both bioinformatics and biochemical analysis have focused on the subunit structure of trypanosome RNA polymerases, this is the first study that reveals a functional RNA polymerase II enzyme.
在布氏锥虫中，RNA聚合酶II的转录负责剪接前导（SL）RNA和大多数蛋白质编码mRNA的表达。为了了解这些寄生虫中RNA聚合酶II转录的调控机制，我们通过对其必需亚基RPB4进行亲和层析，纯化了一种具有转录活性的酶。该酶制剂在非依赖启动子和依赖启动子的体外转录试验中均具有活性。重要的是，该酶对α-鹅膏蕈碱抑制敏感，这是真核RNA聚合酶II酶的一个标志。通过质谱分析，我们鉴定出了布氏锥虫RNA聚合酶II中以前未观察到的RPB12亚基。TbRPB12含有一个保守的CX(2)CX(10 - 15)CX(2)C锌结合基序，这是其他真核RPB12多肽的特征。我们还鉴定出了七种与布氏锥虫RNA聚合酶II相关的蛋白质。虽然生物信息学和生化分析都集中在锥虫RNA聚合酶的亚基结构上，但这是第一项揭示功能性RNA聚合酶II酶的研究。
