MAPPING OF FUNCTIONAL DOMAINS IN EUKARYOTIC PROTEIN-SYNTHESIS INITIATION-FACTOR 4G (EIF4G) WITH PICORNAVIRAL PROTEASES - IMPLICATIONS FOR CAP-DEPENDENT AND CAP-INDEPENDENT TRANSLATIONAL INITIATION
MAPPING OF FUNCTIONAL DOMAINS IN EUKARYOTIC PROTEIN-SYNTHESIS INITIATION-FACTOR 4G (EIF4G) WITH PICORNAVIRAL PROTEASES - IMPLICATIONS FOR CAP-DEPENDENT AND CAP-INDEPENDENT TRANSLATIONAL INITIATION
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DOI：
10.1074/jbc.270.37.21975
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发表时间：
1995-09-15
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：
LAMPHEAR, BJ;KIRCHWEGER, R;RHOADS, RE
翻译起始过程中 mRNA 与 40S 核糖体亚基的帽依赖性结合需要真核起始因子 4G（eIF4G；以前的 eIF-4 gamma 和 p220）与其他起始因子（特别是 eIF4E、eIF4A 和 eIF3）的关联。小核糖核酸病毒感染细胞会导致 eIF4G 蛋白水解并产生不依赖帽的翻译状态。使用鼻病毒 2A 蛋白酶和口蹄疫病毒 L 蛋白酶分析 eIF4G 与 eIF4A、eIF4E 和 eIF3 的关联。两种蛋白酶将 eIF4G 一分为二，称为 cp(N) 和 cp(C) 的 N 端和 C 端片段。通过在 m(7)GTP-Sepharose 上的保留来判断，cp(N) 含有 eIF4E 结合位点，而根据超速离心共沉降，cp(C) 与 eIF3 和 eIF4A 结合。 L 蛋白酶对 cp(N) 的进一步蛋白水解产生了称为 cp(N2) 的 18-kDa 多肽，其保留了 eIF4E 结合活性并对应于兔 eIF4G 的氨基酸残基 319-479。 cp(C) 的进一步蛋白水解产生了几个较小的片段。 cp(C2)（类似于887-1402）包含eIF4A结合位点，而cp(C3)（类似于480-886）包含eIF3结合位点。这些结果表明，小核糖核酸病毒蛋白酶在残基 479-486 处的切割将 eIF4G 分成两个结构域，一个结构域用于募集加帽的 mRNA，另一个结构域用于将 mRNA 连接到核糖体并指导解旋酶活性。只有后者似乎是小核糖核酸病毒 RNA 的内部启动所必需的。
Cap-dependent binding of mRNA to the 40 S ribosomal subunit during translational initiation requires the association of eukaryotic initiation factor 4G (eIF4G; formerly eIF-4 gamma and p220) with other initiation factors, notably eIF4E, eIF4A, and eIF3. Infection of cells by picornaviruses results in proteolytic cleavage of eIF4G and generation of a cap-independent translational state. Rhinovirus 2A protease and foot-and-mouth-disease virus L protease were used to analyze the association of eIF4G with eIF4A, eIF4E, and eIF3. Both proteases bisect eIF4G into N- and C-terminal fragments termed cp(N) and cp(C). cp(N) was shown to contain the eIF4E-binding site, as judged by retention on m(7)GTP-Sepharose, whereas cp(C) was bound to eIF3 and eIF4A, based on ultracentrifugal co-sedimentation. Further proteolysis of cp(N) by L protease produced an 18-kDa polypeptide termed cp(N2) which retained eIF4E binding activity and corresponded to amino acid residues 319-479 of rabbit eIF4G. Further proteolysis of cp(C) yielded several smaller fragments. cp(C2) (similar to 887-1402) contained the eIF4A binding site, whereas cp(C3) (similar to 480-886) contained the eIF3 binding site. These results suggest that cleavage by picornaviral proteases at residues 479-486 separates eIF4G into two domains, one required for recruiting capped mRNAs and one for attaching mRNA to the ribosome and directing helicase activity. Only the latter would appear to be necessary for internal initiation of picornaviral RNAs.