The Gcd10p/Gcd14p complex is the essential two-subunit tRNA(1-methyladenosine) methyltransferase of Saccharomyces cerevisiae
The Gcd10p/Gcd14p complex is the essential two-subunit tRNA(1-methyladenosine) methyltransferase of Saccharomyces cerevisiae
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DOI：
10.1073/pnas.090102597
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发表时间：
2000-05-09
影响因子：
11.1
通讯作者：
Hinnebusch, AG
中科院分区：
文献类型：
--
作者：
Anderson, J;Phan, L;Hinnebusch, AG
修饰的核苷 1-甲基腺苷 (m(1)A) 位于许多真核生物 tRNA 的 T Psi C 环的第 58 位。缺乏 Gcd10p 的酿酒酵母突变体中所有 tRNA 中 m(1)A 的缺失导致起始子蛋氨酸 tRNA (tRNA(i)(Met)) 的加工和稳定性存在严重缺陷。 Gcd10p 与 Gcd14p 形成复合物，后者包含用于结合 S-腺苷甲硫氨酸 (AdoMet) 的保守基序。这些事实，加上我们证明 gcd14 Delta 细胞缺乏 m(1)A，强烈表明 Gcd10p/Gcd14p 复合物是酵母 tRNA(m(1)A) 甲基转移酶 [(m(1)A)MTase]。支持这一预测的是，亲和纯化的 Gcd10p/Gcd14p 复合物使用 AdoMet 作为甲基供体，在总 tRNA 或仅缺少此修饰的纯化 tRNA(i)(Met) 中合成 m(1)A。纯化复合物的动力学分析显示AdoMet或tRNA(i)(Met)的KM值分别为5.0μM和2.5nM。预测的 AdoMet 结合结构域的突变破坏了体内 GCD14 功能和体外 (m(1)A)MTase 活性。纯化的带有 Flag 标签的 Gcd14p 单独没有酶活性，与野生型复合物相比，tRNA 结合严重受损，表明 Gcd10p 是 tRNA 底物紧密结合所必需的。我们的结果证明了双组分 tRNA MTase，并表明 AdoMet 和 tRNA 底物的结合取决于复合物的不同亚基。
The modified nucleoside 1-methyladenosine (m(1)A) is found at position 58 in the T Psi C loop of many eukaryotic tRNAs. The absence of m(1)A from ail tRNAs in Saccharomyces cerevisiae mutants lacking Gcd10p elicits severe defects in processing and stability of initiator methionine tRNA (tRNA(i)(Met)). Gcd10p is found in a complex with Gcd14p, which contains conserved motifs for binding S-adenosyl-methionine (AdoMet). These facts, plus our demonstration that gcd14 Delta cells lacked m(1)A, strongly suggested that Gcd10p/Gcd14p complex is the yeast tRNA(m(1)A)methyltransferase [(m(1)A)MTase]. Supporting this prediction, affinity-purified Gcd10p/Gcd14p complexes used AdoMet as a methyl donor to synthesize m(1)A in either total tRNA or purified tRNA(i)(Met) lacking only this modification. Kinetic analysis of the purified complex revealed KM values for AdoMet or tRNA(i)(Met) Of 5.0 mu M and 2.5 nM, respectively. Mutations in the predicted AdoMet-binding domain destroyed GCD14 function in vivo and (m(1)A)MTase activity in vitro. Purified Flag-tagged Gcd14p alone had no enzymatic activity and was severely impaired for tRNA-binding compared with the wild-type complex, suggesting that Gcd10p is required for tight binding of the tRNA substrate. Our results provide a demonstration of a two-component tRNA MTase and suggest that binding of AdoMet and tRNA substrates depends on different subunits of the complex.