Walgraffe David, Devaux Sara, Lecordier Laurence, Dierick Jean-François, Dieu Marc, Van den Abbeele Jan, Pays Etienne, Vanhamme Luc
Laboratory of Molecular Parasitology, Department of Molecular Biology, IBMM, Free University of Brussels, 12, rue des Professeurs Jeener et Brachet, B-6041 Gosselies, Belgium.
Mol Biochem Parasitol. 2005 Feb;139(2):249-60. doi: 10.1016/j.molbiopara.2004.11.014.
The Trypanosoma brucei homologue of the RNA polymerase I (RNA Pol I) subunit Rpa12p of Saccharomyces cerevisiae was cloned and characterized. This protein did not appear to be essential for growth in either bloodstream or procyclic forms of the parasite. Trypanosomes expressing a C-terminal tagged version of TbRPA12 were generated in order to purify RNA Pol I from both developmental stages. Tandem affinity purification (TAP) revealed a number of proteins associating with TbRPA12, some of which appeared to be stage-specific. Mass spectrometry allowed the identification of four subunits in addition to TbRPA12, namely TbRPA1, TbRPA2, TbRPC40 and one isoform of TbRPB5 (Tb1RPB5), as well as an unknown 30kDa protein and histones H2A and H3. Whereas these studies demonstrated that TbRPA1 was phosphorylated, no evidence for phosphorylation of TbRPA2 was found.
克隆并鉴定了酿酒酵母RNA聚合酶I（RNA Pol I）亚基Rpa12p的布氏锥虫同源物。该蛋白对于寄生虫的血流形式或前循环形式的生长似乎并非必需。为了从两个发育阶段纯化RNA Pol I，构建了表达C端标记的TbRPA12的锥虫。串联亲和纯化（TAP）揭示了许多与TbRPA12相关的蛋白质，其中一些似乎具有阶段特异性。质谱分析除了鉴定出TbRPA12外，还鉴定出四个亚基，即TbRPA1、TbRPA2、TbRPC40和TbRPB5的一种同工型（Tb1RPB5），以及一种未知的30kDa蛋白和组蛋白H2A和H3。虽然这些研究表明TbRPA1被磷酸化，但未发现TbRPA2磷酸化的证据。
