Peris M, Simpson A M, Grunstein J, Liliental J E, Frech G C, Simpson L
Department of Biology, University of California, Los Angeles 90095-662, USA.
Mol Biochem Parasitol. 1997 Mar;85(1):9-24. doi: 10.1016/s0166-6851(96)02795-8.
Two polypeptides of 50 and 45 kDa were adenylated by incubation of a mitochondrial extract from Leishmania tarentolae with [alpha-32P]ATP. These proteins were components of a complex that sedimented at 20S in glycerol gradients and migrated as a single band of approximately 1800 kDa in a native gel. The facts that RNA ligase activity cosedimented at 20S and that the ATP-labeled p45 and p50 polypeptides were deadenylated upon incubation with a ligatable RNA substrate suggested that these proteins may represent charged intermediates of a mitochondrial RNA ligase. Hybridization of native gel blots with guide RNA (gRNA) probes showed the presence of gRNA in the previously identified T-IV complexes that sedimented in glycerol at 10S and contained terminal uridylyl transferase (TUTase) activity, and also in a previously unidentified class of heterodisperse complexes that sedimented throughout the gradient. gRNAs were not detected in the p45 + p50-containing 1800 kDa complex. The heterodisperse gRNA-containing complexes were sensitive to incubation at 27 degrees C and appear to represent complexes of T-IV subunits with mRNA. Polyclonal antiserum to a 70 kDa protein that purified with terminal uridylyl transferase activity was generated, and the antiserum was used to show that this p70 polypeptide was a component of both the T-IV and the heterodisperse gRNA-containing complexes. We propose that the p45 + p50-containing 1800 kDa complex and the p70 + gRNA-containing heterodisperse complexes interact in the editing process. Further characterization of these various complexes should increase our knowledge of the biochemical mechanisms involved in RNA editing.
通过将来自热带利什曼原虫的线粒体提取物与[α-32P]ATP一起温育，使50 kDa和45 kDa的两种多肽发生腺苷酸化。这些蛋白质是一种复合物的组分，该复合物在甘油梯度中以20S沉降，并在天然凝胶中迁移为一条约1800 kDa的单一带。RNA连接酶活性在20S处共沉降，并且ATP标记的p45和p50多肽在与可连接的RNA底物温育时去腺苷酸化，这些事实表明这些蛋白质可能代表线粒体RNA连接酶的带电荷中间体。用引导RNA（gRNA）探针与天然凝胶印迹杂交显示，在先前鉴定的T-IV复合物中存在gRNA，该复合物在甘油中以10S沉降并具有末端尿苷酰转移酶（TUTase）活性，并且还存在于先前未鉴定的一类在整个梯度中沉降的异源分散复合物中。在含有p45 + p50的1800 kDa复合物中未检测到gRNA。含有异源分散gRNA的复合物在27℃温育时敏感，似乎代表T-IV亚基与mRNA的复合物。产生了针对具有末端尿苷酰转移酶活性纯化的70 kDa蛋白质的多克隆抗血清，并且该抗血清用于表明该p70多肽是T-IV和含有异源分散gRNA的复合物的组分。我们提出，含有p45 + p50的1800 kDa复合物和含有p70 + gRNA的异源分散复合物在编辑过程中相互作用。对这些各种复合物的进一步表征应会增加我们对RNA编辑所涉及的生化机制的了解。
