Interaction between the NH2-terminal domain of eIF4A and the central domain of eIF4G modulates RNA-stimulated ATPase activity
Interaction between the NH2-terminal domain of eIF4A and the central domain of eIF4G modulates RNA-stimulated ATPase activity
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DOI：
10.1074/jbc.m406168200
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发表时间：
2005-01-21
影响因子：
4.8
通讯作者：
Rhoads, RE
中科院分区：
文献类型：
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作者：
Korneeva, NL;First, EA;Rhoads, RE
真核细胞翻译因子4A(EIF4A)是DEA(D/H)-box RNA解旋酶家族的成员之一，它是一类将ATP水解与RNA结合和双链分离偶联的蛋白质。EIF4A通过解开mRNAs 5‘-非翻译区的二级结构，促进40个S核糖体亚基对起始密码子的扫描，参与了翻译的启动。EIF4A单独只有微弱的ATPase和解旋酶活性，但它们被eIF4G、eIF4B和eIF4H刺激。EIF4G有两个eIF4A结合位点，一个在中央结构域(cp(C3))，另一个在COOH-末端结构域(cp(C2))。在目前的工作中，我们证明了这两个eIF4G结构域对RNA刺激的eIF4A的ATPase活性具有不同的影响。CP(C3)通过两种机制使ATP水解率提高约40倍：K-m RNA降低10倍和K(CAT)升高4倍。CP(C3)还以不依赖于ATP的方式刺激RNA与eIF4A的交联。对eIF4G和eIF4A变体的研究表明，cp(C3)通过改变催化部位的构象来促进RNA结合的模型。CP(C2)不刺激ATPase活性，并进一步增加K-m(ATP)(在饱和RNA浓度下)和K-m(RNA)(在亚饱和浓度下)。Cp(C3)和cp(C2)都直接与eIF4A的NH2-末端结构域相互作用，该结构域具有保守的ATP和寡核苷酸结合基序，但不与COOH-末端结构域相互作用。
The eukaryotic translation factor 4A (eIF4A) is a member of DEA(D/H)-box RNA helicase family, a diverse group of proteins that couples ATP hydrolysis to RNA binding and duplex separation. eIF4A participates in the initiation of translation by unwinding secondary structure in the 5'-untranslated region of mRNAs and facilitating scanning by the 40 S ribosomal subunit for the initiation codon. eIF4A alone has only weak ATPase and helicase activities, but these are stimulated by eIF4G, eIF4B, and eIF4H. eIF4G has two eIF4A-binding sites, one in the central domain (cp(C3)) and one in the COOH-terminal domain (cp(C2)). In the current work, we demonstrate that these two eIF4G domains have different effects on the RNA-stimulated ATPase activity of eIF4A. cp(C3) stimulates ATP-hydrolytic efficiency by about 40-fold through two mechanisms: lowering K-m RNA by 10-fold and raising k(cat) by 4-fold. cp(C3) also stimulates RNA cross-linking to eIF4A in an ATP-independent manner. Studies with eIF4G and eIF4A variants suggest a model by which cp(C3) alters the conformation of the catalytic site to favor RNA binding. cp(C2) does not stimulate ATPase activity and furthermore increases both K-m (ATP) (at saturating RNA concentrations) and K-m (RNA) ( at subsaturating ATP concentrations). Both cp(C3) and cp(C2) directly interact with the NH2-terminal domain of eIF4A, which possesses conserved ATP- and oligonucleotide-binding motifs, but not with the COOH-terminal domain.