Blum S, Schmid S R, Pause A, Buser P, Linder P, Sonenberg N, Trachsel H
Institut für Biochemie und Molekularbiologie, Universität Bern, Switzerland.
Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7664-8. doi: 10.1073/pnas.89.16.7664.
Saccharomyces cerevisiae translation initiation factor eIF-4A, an RNA helicase of the Asp-Glu-Ala-Asp (DEAD) box protein family, was mutated in the putative ATP binding site and expressed in Escherichia coli. Mutant proteins with alanine at position 66 replaced by glycine [eIF-4A(A66G)] or valine [eIF-4A(A66V)] were purified from Escherichia coli extracts and analyzed in vitro for activity in ATP crosslinking, ATP hydrolysis, RNA helicase, and translation assays. The results show that in vitro ATP hydrolysis activity, RNA helicase activity, and translation activity of eIF-4A correlate with in vivo activity of the factor. Whereas eIF-4A(A66G) showed wild-type activity in all assays, eIF-4A(A66V) was active in ATP crosslinking but inactive in ATP hydrolysis and RNA helicase assays. In vitro translation was supported by wild-type eIF-4A and eIF-4A(A66G) but not by eIF-4A(A66V). The results show that, for their translation, the majority of mRNAs from Saccharomyces cerevisiae including an mRNA with the initiator AUG positioned 8 nucleotides downstream of the cap structure require eIF-4A that is able to hydrolyze ATP.
酿酒酵母翻译起始因子eIF - 4A是天冬氨酸 - 谷氨酸 - 丙氨酸 - 天冬氨酸（DEAD）盒蛋白家族的一种RNA解旋酶，其在假定的ATP结合位点发生突变，并在大肠杆菌中表达。从大肠杆菌提取物中纯化出第66位丙氨酸被甘氨酸取代的突变蛋白[eIF - 4A(A66G)]或被缬氨酸取代的突变蛋白[eIF - 4A(A66V)]，并在体外对其进行ATP交联、ATP水解、RNA解旋酶和翻译测定的活性分析。结果表明，eIF - 4A的体外ATP水解活性、RNA解旋酶活性和翻译活性与该因子的体内活性相关。虽然eIF - 4A(A66G)在所有测定中均表现出野生型活性，但eIF - 4A(A66V)在ATP交联中具有活性，而在ATP水解和RNA解旋酶测定中无活性。体外翻译由野生型eIF - 4A和eIF - 4A(A66G)支持，但不由eIF - 4A(A66V)支持。结果表明，对于酿酒酵母的大多数mRNA（包括起始AUG位于帽结构下游8个核苷酸处的mRNA）的翻译而言，需要能够水解ATP的eIF - 4A。
