Kinetoplastid mRNAs possess a unique hypermethylated cap 4 structure derived from the standard m7GpppN cap structure, with 2'-O methylations on the first four ribose sugars and additional base methylations on the first adenine and the fourth uracil. While the enzymes responsible for m7GpppN cap 0 formations has been characterized in Trypanosoma brucei, the mechanism of cap 4 methylation and the role of the hypermethylated structure remain unclear. Here, we describe the characterization of a 48 kDa T.brucei 2'-O nucleoside methyltransferase (TbCom1). Recombinant TbCom1 transfers the methyl group from S-adenosylmethionine (AdoMet) to the 2'-OH of the second nucleoside of m7GpppNpNp-RNA to form m7GpppNpNmp-RNA. TbCom1 is also capable of converting cap 1 RNA to cap 2 RNA. The methyl transfer reaction is dependent on the m7GpppN cap, as the enzyme does not form a stable interaction with GpppN-terminated RNA. Mutational analysis establishes that the TbCom1 and vaccinia virus VP39 methyltransferases share mechanistic similarities in AdoMet- and cap-recognition. Two aromatic residues, Tyr18 and Tyr187, may participate in base-stacking interactions with the guanine ring of the cap, as the removal of each of these aromatic side-chains abolishes cap-specific RNA-binding.

译文

kinetopplastid mrna具有源自标准m7GpppN cap结构的独特的超甲基化cap 4结构,在前四个核糖上具有2 '-O甲基化,在第一个腺嘌呤和第四个尿嘧啶上具有其他碱基甲基化。尽管在布氏锥虫中已经表征了负责m7GpppN cap 0形成的酶,但cap 4甲基化的机制和超甲基化结构的作用仍不清楚。在这里,我们描述了48 kDa T.br ucei 2 '-O核苷甲基转移酶 (TbCom1) 的表征。重组TbCom1将甲基从S-腺苷甲硫氨酸 (AdoMet) 转移至m7GpppNpNp-RNA第二核苷的2 '-OH形成m7GpppNpNmp-RNA。TbCom1还能够将cap 1 RNA转换为cap 2 RNA。甲基转移反应取决于m7GpppN帽,因为该酶不会与GpppN终止的RNA形成稳定的相互作用。突变分析表明,TbCom1和痘苗病毒VP39甲基转移酶在AdoMet和cap识别中具有机械相似性。两个芳香族残基Tyr18和Tyr187可能参与与cap的鸟嘌呤环的碱基堆积相互作用,因为这些芳香族侧链中的每一个的去除消除了cap特异性RNA的结合。

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