Unexpected accumulation of ncm(5)U and ncm(5)S(2) (U) in a trm9 mutant suggests an additional step in the synthesis of mcm(5)U and mcm(5)S(2)U.
Unexpected accumulation of ncm(5)U and ncm(5)S(2) (U) in a trm9 mutant suggests an additional step in the synthesis of mcm(5)U and mcm(5)S(2)U.
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DOI：
10.1371/journal.pone.0020783
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Byström AS
中科院分区：
文献类型：
--
作者：
Chen C;Huang B;Anderson JT;Byström AS
转移 RNA 作为初级转录物合成，经过加工产生成熟的 tRNA。作为成熟过程的一部分，核苷的子集被修饰。反密码子区域的修饰通常会调节 tRNA 的解码能力。在第 34 位，大多数具有尿苷的酵母胞质 tRNA 物种被修饰为 5-氨基甲酰甲基尿苷 (ncm5U)、5-氨基甲酰甲基-2'-O-甲基尿苷 (ncm5Um)、5-甲氧基羰基甲基-尿苷 (mcm5U) 或 5-甲氧基羰基甲基-2-硫尿苷 (mcm5s2U)。 mcm5和ncm5侧链的形成涉及一个复杂的途径，其中mcm5形成的最后一步是依赖于Trm9和Trm112蛋白的cm5的甲酯化。在摆动尿苷处形成 mcm5 侧链的最后一步需要 Trm9 和 Trm112。通过在大肠杆菌中共表达组氨酸标记的 Trm9p 和天然 Trm112p，这两种蛋白质被纯化为复合物。 Trm112p 的存在显着提高了 Trm9p 的体外甲基转移酶活性。从 trm9Δ 或 trm112Δ 突变体中分离出通常含有 mcm5U 或 mcm5s2U 核苷的单一 tRNA 种类，并通过 HPLC 分析修饰核苷的存在。在这两种突变体中，tRNA 中均不存在 mcm5U 和 mcm5s2U 核苷，累积的主要中间体是 ncm5U 和 ncm5s2U，而不是预期的 cm5U 和 cm5s2U。 Trm9p 和 Trm112p 在使用中间 cm5U 作为底物在 tRNA 中形成 mcm5U 的最后一步中共同发挥作用。在从 trm9Δ 和 trm112Δ 菌株分离的 tRNA 中，ncm5U 和 ncm5s2U 核苷积累，质疑 mcm5 侧链核苷中间体形成的顺序。我们对这一观察结果提出了两种替代解释。一种是中间cm5U是由ncm5U通过未知机制生成的，另一种是cm5U是在ncm5U和mcm5U之前形成的。
Transfer RNAs are synthesized as a primary transcript that is processed to produce a mature tRNA. As part of the maturation process, a subset of the nucleosides are modified. Modifications in the anticodon region often modulate the decoding ability of the tRNA. At position 34, the majority of yeast cytosolic tRNA species that have a uridine are modified to 5-carbamoylmethyluridine (ncm5U), 5-carbamoylmethyl-2′-O-methyluridine (ncm5Um), 5-methoxycarbonylmethyl-uridine (mcm5U) or 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). The formation of mcm5 and ncm5 side chains involves a complex pathway, where the last step in formation of mcm5 is a methyl esterification of cm5 dependent on the Trm9 and Trm112 proteins. Both Trm9 and Trm112 are required for the last step in formation of mcm5 side chains at wobble uridines. By co-expressing a histidine-tagged Trm9p together with a native Trm112p in E. coli, these two proteins purified as a complex. The presence of Trm112p dramatically improves the methyltransferase activity of Trm9p in vitro. Single tRNA species that normally contain mcm5U or mcm5s2U nucleosides were isolated from trm9Δ or trm112Δ mutants and the presence of modified nucleosides was analyzed by HPLC. In both mutants, mcm5U and mcm5s2U nucleosides are absent in tRNAs and the major intermediates accumulating were ncm5U and ncm5s2U, not the expected cm5U and cm5s2U. Trm9p and Trm112p function together at the final step in formation of mcm5U in tRNA by using the intermediate cm5U as a substrate. In tRNA isolated from trm9Δ and trm112Δ strains, ncm5U and ncm5s2U nucleosides accumulate, questioning the order of nucleoside intermediate formation of the mcm5 side chain. We propose two alternative explanations for this observation. One is that the intermediate cm5U is generated from ncm5U by a yet unknown mechanism and the other is that cm5U is formed before ncm5U and mcm5U.
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影响因子：
64.8
作者：
Gavin, AC;Bösche, M;Superti-Furga, G
通讯作者：
Superti-Furga, G
影响因子：
64.8
作者：
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影响因子：
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5.3
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5.3
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