Devaux Sara, Lecordier Laurence, Uzureau Pierrick, Walgraffe David, Dierick Jean-François, Poelvoorde Philippe, Pays Etienne, Vanhamme Luc
Laboratory of Molecular Parasitology, Institute for Molecular Biology and Medicine (IBMM), Université Libre de Bruxelles, Gosselies, Belgium.
Mol Biochem Parasitol. 2006 Jul;148(1):60-8. doi: 10.1016/j.molbiopara.2006.02.020. Epub 2006 Mar 23.
The Trypanosoma brucei homolog of the RNA polymerase II (RNA Pol II) subunit RPB9 was cloned and characterized. Contrary to what occurs in Saccharomyces cerevisiae, in T. brucei this protein was found to be essential since the knock down of its expression by RNAi led to lethality in both bloodstream and procyclic forms of the parasite. As expected, TbRPB9 knock down specifically inhibited transcription by RNA Pol II, but not by RNA Pol I and III. TbRPB9 was used as bait to isolate the RNA Pol II core complex by tandem affinity purification. Nine subunits homologous to the other eukaryotic RNA Pol II, namely RPB1, RPB2, RPB3, RPB4, RPB5, RPB6, RPB7, RPB8 and RPB11, were identified in the purified complex. Interestingly, the RPB5 homolog associated with RNA Pol II was different from the one previously found in RNA Pol I. Analysis of the genome database revealed the presence of genes for all purified subunits plus RPB10. As in the case of TbRPB5, two genes coding for different isoforms of TbRPB6 were identified, suggesting the existence of polymerase-specific isoforms for both TbRPB5 and TbRPB6.
克隆并鉴定了RNA聚合酶II（RNA Pol II）亚基RPB9的布氏锥虫同源物。与酿酒酵母中的情况相反，在布氏锥虫中发现该蛋白是必需的，因为通过RNA干扰敲低其表达会导致寄生虫的血流形式和前循环形式均致死。正如预期的那样，敲低TbRPB9会特异性抑制RNA Pol II的转录，但不会抑制RNA Pol I和III的转录。通过串联亲和纯化，将TbRPB9用作诱饵来分离RNA Pol II核心复合物。在纯化的复合物中鉴定出了与其他真核生物RNA Pol II同源的九个亚基，即RPB1、RPB2、RPB3、RPB4、RPB5、RPB6、RPB7、RPB8和RPB11。有趣的是，与RNA Pol II相关的RPB5同源物与先前在RNA Pol I中发现的不同。对基因组数据库的分析揭示了所有纯化亚基以及RPB10的基因的存在。与TbRPB5的情况一样，鉴定出了编码TbRPB6不同同工型的两个基因，这表明TbRPB5和TbRPB6都存在聚合酶特异性同工型。
