Darzynkiewicz E, Stepinski J, Ekiel I, Goyer C, Sonenberg N, Temeriusz A, Jin Y, Sijuwade T, Haber D, Tahara S M
Department of Microbiology, University of Southern California School of Medicine, Los Angeles 90033-1054.
Biochemistry. 1989 May 30;28(11):4771-8. doi: 10.1021/bi00437a038.
Nucleotide cap analogues of 7-methylguanosine 5'-monophosphate (m7GMP) were synthesized in which the 7-methyl moiety was replaced with 7-ethyl (e7), 7-propyl (p7), 7-isopropyl (ip7), 7-butyl (b7), 7-isobutyl (ib7), 7-cyclopentyl (cp7), 7-(carboxymethyl) (cm7), 7-benzyl (bn7), 7-(2-phenylethyl) [7-(2-PhEt)], and 7-(1-phenylethyl) [7-(1-PhEt)]. These derivatives were assayed as competitive inhibitors of capped mRNA translation in reticulocyte lysate. We observed that N7 alkyl and alicyclic substituents larger than ethyl significantly decreased the inhibitory activity of these cap analogues presumably by decreasing their affinity for cap binding proteins, which participate in the initiation of translation. This result defined a maximum size for this class of N7 substituents in the nucleotide binding domain of cap binding proteins. Like m7GMP, the N7-substituted GMP derivatives synthesized in this study were found to be predominantly in the anti conformation as determined by proton NMR analyses. However, bn7GMP and 7-(2-PhEt)GMP, which have aromatic N7 substituents, were more effective than m7GMP as competitive inhibitors of translation. The increased affinity of bn7GMP for cap binding proteins was further examined by synthesis of beta-globin mRNA containing 5'-bn7G, 5'-m7G, or 5'-e7G cap structures. These modified mRNAs were tested as translation templates. Messenger RNA capped with bn7G was observed to increase the translation activity of the template 1.8-fold relative to that of its m7G-capped mRNA counterpart. By contrast, e7G-capped mRNA was 25% less active than m7G-capped mRNA.2+V photo-cross-linking of m7G-capped mRNA to cap binding proteins
合成了7-甲基鸟苷5'-单磷酸（m7GMP）的核苷酸帽类似物，其中7-甲基部分被7-乙基（e7）、7-丙基（p7）、7-异丙基（ip7）、7-丁基（b7）、7-异丁基（ib7）、7-环戊基（cp7）、7-（羧甲基）（cm7）、7-苄基（bn7）、7-（2-苯乙基）[7-（2-PhEt）]和7-（1-苯乙基）[7-（1-PhEt）]取代。这些衍生物作为网织红细胞裂解物中加帽mRNA翻译的竞争性抑制剂进行了测定。我们观察到，大于乙基的N7烷基和脂环族取代基显著降低了这些帽类似物的抑制活性，推测是通过降低它们与参与翻译起始的帽结合蛋白的亲和力。这一结果确定了帽结合蛋白核苷酸结合域中此类N7取代基的最大尺寸。与m7GMP一样，通过质子核磁共振分析确定，本研究中合成的N7取代的GMP衍生物主要处于反式构象。然而，具有芳香族N7取代基的bn7GMP和7-（2-PhEt）GMP作为翻译的竞争性抑制剂比m7GMP更有效。通过合成含有5'-bn7G、5'-m7G或5'-e7G帽结构的β-珠蛋白mRNA，进一步研究了bn7GMP对帽结合蛋白的增强亲和力。这些修饰的mRNA作为翻译模板进行了测试。观察到用bn7G加帽的信使RNA相对于其m7G加帽的mRNA对应物，模板的翻译活性提高了1.8倍。相比之下，e7G加帽的mRNA活性比m7G加帽的mRNA低25%。m7G加帽的mRNA与帽结合蛋白的2+V光交联
