The tRNA-interacting factor p43 associates with mammalian arginyl-tRNA synthetase but does not modify its tRNA aminoacylation properties
The tRNA-interacting factor p43 associates with mammalian arginyl-tRNA synthetase but does not modify its tRNA aminoacylation properties
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DOI：
10.1021/bi036150e
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发表时间：
2004-04-20
期刊：
影响因子：
2.9
通讯作者：
Mirande, M
中科院分区：
文献类型：
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作者：
Guigou, L;Shalak, V;Mirande, M
精氨酰-tRNA合成酶（ArgRS）是一种多酶复合物的九种合成酶组分之一，也含有三种辅助蛋白。我们以前建立了辅助蛋白p43的N-末端部分与ArgRS的N-末端真核生物特异性多肽延伸相关联。由于p43与Arc 1 p同源，Arc 1 p是一种酵母通用RNA结合蛋白，与MetRS和GluRS相关，并在氨酰化反应中发挥tRNA结合辅因子的作用，我们分析了p43-ArgRS关联的功能意义。我们先前已经表明，全长ArgRS（对应于多合成酶复合物内相关的ArgRS种类）和缺失73个N-末端氨基酸残基的ArgRS（对应于ArgRS的游离种类，两者都在酵母中产生）具有相似的催化参数（Lazard，M.，Kerjan，P.，Agou，F.，和Mirande，M.等人（2000）J. Mol. 302，991-1004）。然而，最近的一项研究表明，p43与ArgRS的结合降低了ArgRS对tRNA的表观Km（Park，S. G.，荣格，K. H、李，J.S.，Jo，Y. J.，莫泰吉，H.，Kim，S.，和Shiba，K.（1999）J.Biol.Chem.274，16673-16676）。在这项研究中，我们详细分析，凝胶阻滞试验和酶动力学，p43作为tRNA结合辅因子的ArgRS的推定作用。p43与ArgRS的关联既不增强tRNA结合，也不改变氨基酸活化或tRNA氨酰化反应中的动力学参数。此外，选择性去除的C-末端RNA结合结构域的p43从多合成酶复合物不影响ArgRS的动力学参数。因此，p43具有双重功能。它通过其N末端结构域促进ArgRS与复合物的结合，但其C末端RNA结合结构域可能充当复合物中尚未鉴定的成分的tRNA相互作用因子。
Arginyl-tRNA synthetase (ArgRS) is one of the nine synthetase components of a multienzyme complex containing three auxiliary proteins as well. We previously established that the N-terminal moiety of the auxiliary protein p43 associates with the N-terminal, eukaryotic-specific polypeptide extension of ArgRS. Because p43 is homologous to Arc1p, a yeast general RNA-binding protein that associates with MetRS and GluRS and plays the role of tRNA-binding cofactor in the aminoacylation reaction, we analyzed the functional significance of p43-ArgRS association. We had previously showed that full-length ArgRS, corresponding to the ArgRS species associated within the multisynthetase complex, and ArgRS with a deletion of 73 N-terminal amino acid residues, corresponding to a free species of ArgRS, both produced in yeast, have similar catalytic parameters (Lazard, M., Kerjan, P., Agou, F., and Mirande, M. (2000) J. Mol. Biol. 302, 991-1004). However, a recent study had suggested that association of p43 to ArgRS reduces the apparent Km of ArgRS to tRNA (Park, S. G., Jung, K. H., Lee, J. S., Jo, Y. J., Motegi, H., Kim, S., and Shiba, K. (1999) J. Biol. Chem. 274, 16673-16676). In this study, we analyzed in detail, by gel retardation assays and enzyme kinetics, the putative role of p43 as a tRNA-binding cofactor of ArgRS. The association of p43 with ArgRS neither strengthened tRNA-binding nor changed kinetic parameters in the amino acid activation or in the tRNA aminoacylation reaction. Furthermore, selective removal of the C-terminal RNA-binding domain of p43 from the multisynthetase complex did not affect kinetic parameters for ArgRS. Therefore, p43 has a dual function. It promotes association of ArgRS to the complex via its N-terminal domain, but its C-terminal RNA-binding domain may act as a tRNA-interacting factor for an as yet unidentified component of the complex.