INHIBITION OF EUKARYOTIC TRANSLATION BY NUCLEOSIDE 5'-MONOPHOSPHATE ANALOGS OF MESSENGER-RNA 5'-CAP - CHANGES IN N7 SUBSTITUENT AFFECT ANALOG ACTIVITY
INHIBITION OF EUKARYOTIC TRANSLATION BY NUCLEOSIDE 5'-MONOPHOSPHATE ANALOGS OF MESSENGER-RNA 5'-CAP - CHANGES IN N7 SUBSTITUENT AFFECT ANALOG ACTIVITY
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DOI：
10.1021/bi00437a038
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发表时间：
1989-05-30
期刊：
影响因子：
2.9
通讯作者：
TAHARA, SM
中科院分区：
文献类型：
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作者：
DARZYNKIEWICZ, E;STEPINSKI, J;TAHARA, SM
合成了7-甲基鸟苷5“-单磷酸（m7 GMP）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（ib 7）、7-环戊基（cp 7）、7-异丙基（p7）（羧甲基）（cm 7）、7-苄基（bn 7）、7-（2-苯基乙基）[7-（2-PhEt）]和7-（1-苯基乙基）[7-（1-PhEt）]。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测定。我们观察到，N7的烷基和脂环取代基大于乙基显着降低这些帽类似物的抑制活性，大概是通过降低他们的亲和力帽结合蛋白，参与启动翻译。该结果定义了帽结合蛋白的核苷酸结合结构域中这类N7取代基的最大尺寸。像m7 GMP，N7-取代的GMP衍生物在这项研究中合成的被发现主要是在反构象，通过质子NMR分析确定。然而，bn 7 GMP和7-（2-PhEt）GMP，其中有芳香族N7取代基，是更有效的比m7 GMP作为竞争性抑制剂的翻译。bn 7 GMP对帽结合蛋白的增加的亲和力通过合成β-含有5“-bn 7 G、5”-m7 G或5“-e7 G帽结构的球蛋白mRNA。将这些修饰的mRNA作为翻译模板进行测试。观察到用bn 7 G加帽的信使RNA相对于其m7 G加帽的mRNA对应物的翻译活性增加模板的翻译活性1.8倍。相比之下，e7 G加帽的mRNA比m7 G加帽的mRNA活性低25%。UV光交联的m7 G-加帽的mRNA帽结合蛋白也抑制bn 7 GMP在更大程度上比m7 GMP或e7 GMP。因此，根据这些数据，bn 7 GMP的抑制作用是由于其对帽结合蛋白的亲和力增加，而不是由于在另一个起始步骤的抑制。
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. UV photo-cross-linking of m7G-capped mRNA to cap binding proteins was also inhibited to a greater extent by bn7GMP than by m7GMP or e7GMP. Thus, from these data the inhibitory effect of bn7GMP was due to its increased affinity for cap binding proteins and not by inhibition at another step of initiation.