Teraoka Y, Morino S, Tomoo K, Ishida T
Department of Physical Chemistry, Osaka University of Pharmaceutical Sciences, Japan.
Biochem Biophys Res Commun. 1996 Nov 21;228(3):704-8. doi: 10.1006/bbrc.1996.1720.
To investigate the role of four Cys residues of eukaryotic initiation factor (eIF)-4E in the recognition of the mRNA cap structure, single, double and quadruple mutant genes which encoded the Ala residues in the place of the respective Cys residues were prepared using a synthetic human eIF-4E gene by the site-directed mutagenesis, and were expressed in E. coli with the same way as the wild type. The cap binding abilities of respective mutated eIF-4Es were compared with that of the wild-type by m7GTP affinity chromatography. The results suggest that, although all four of Cys residues participate in the recognition of the mRNA cap structure, they contribute indirectly to the stabilization of overall tertiary structure, especially of the cap binding pocket, rather than by direct interaction. A comparison among the cap binding abilities of single, double and quadruple mutants indicated no existence of internal disulfide bonds in eIF-4E.
为了研究真核生物起始因子（eIF）-4E的四个半胱氨酸残基在识别mRNA帽结构中的作用，我们使用合成的人eIF-4E基因通过定点诱变制备了分别编码丙氨酸残基取代相应半胱氨酸残基的单突变、双突变和四突变基因，并以与野生型相同的方式在大肠杆菌中表达。通过m7GTP亲和层析将各个突变的eIF-4E的帽结合能力与野生型进行比较。结果表明，虽然所有四个半胱氨酸残基都参与了mRNA帽结构的识别，但它们是间接有助于整体三级结构的稳定，尤其是帽结合口袋的稳定，而非通过直接相互作用。单突变、双突变和四突变体的帽结合能力比较表明，eIF-4E中不存在内部二硫键。
