Odon O W, Kramer G, Henderson A B, Pinphanichakarn P, Hardesty B
J Biol Chem. 1978 Mar 25;253(6):1807-16.
Three lines of evidence are presented indicating that GTP hydrolysis associated with eukaryotic peptide initiation occurs in the absence of 60 S subunits when methionyl-tRNAf is bound to 40 S ribosomal subunits. An enzyme fraction required for binding of methionyl-tRNAf to 40 S subunits and peptide initiation, tentatively equated with eIF-(4 + 5), has GTPase activity and appears to be responsible for hydrolysis of GTP in the methionyl-tRNAf.eIF-2.GTP complex. Direct analysis of the methionyl-tRNAf.40 S complex formed with with eIF-2 and [8-3H] guanine, [gamma-32P]GTP reveals bound guanine but not gamma-phosphate. Edeine, a peptide antibiotic containing spermidine and beta-tyrosine residues at its COOH terminus and NH2 terminus, respectively, blocks peptide initiation and interferes with binding of methionyl-tRNAf to 40 S ribosomal subunits. Inhibition of binding is observed when the eIF-2-mediated binding reaction is carried out with GTP but not with guanosine 5'-(beta,gamma-methylene)triphosphate or guanosine 5'-(beta,gamma-imido)triphosphate. Edeine was labeled by iodination and shown to bind with high affinity to 40 S but not to 60 S ribosomal subunits. It is suggested that edeine blocks a specific site on the 40 S ribosomal subunit to which a segment of the methionyl-tRNAf molecule is bound during the course of the initiation reaction sequence.
本文提供了三条证据，表明当甲硫氨酰 - tRNAf与40 S核糖体亚基结合时，与真核生物肽起始相关的GTP水解在没有60 S亚基的情况下发生。一种将甲硫氨酰 - tRNAf与40 S亚基结合以及肽起始所需的酶组分，暂定为eIF-(4 + 5)，具有GTPase活性，似乎负责甲硫氨酰 - tRNAf.eIF-2.GTP复合物中GTP的水解。用eIF-2和[8-3H]鸟嘌呤、[γ-32P]GTP形成的甲硫氨酰 - tRNAf.40 S复合物的直接分析显示有结合的鸟嘌呤，但没有γ-磷酸。放线菌素是一种在其COOH末端和NH2末端分别含有亚精胺和β-酪氨酸残基的肽抗生素，它阻断肽起始并干扰甲硫氨酰 - tRNAf与40 S核糖体亚基的结合。当用GTP而不是5'-(β,γ-亚甲基)三磷酸鸟苷或5'-(β,γ-亚氨基)三磷酸鸟苷进行eIF-2介导的结合反应时，观察到结合受到抑制。放线菌素经碘化标记后显示与40 S核糖体亚基有高亲和力结合，而与60 S核糖体亚基无结合。有人提出，放线菌素阻断了40 S核糖体亚基上的一个特定位点，在起始反应序列过程中甲硫氨酰 - tRNAf分子的一段与之结合。
