Specific functional interactions of nucleotides at key -3 and +4 positions flanking the initiation codon with components of the mammalian 48S translation initiation complex
Specific functional interactions of nucleotides at key -3 and +4 positions flanking the initiation codon with components of the mammalian 48S translation initiation complex
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DOI：
10.1101/gad.1397906
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发表时间：
2006-03-01
影响因子：
10.5
通讯作者：
Pestova, TV
中科院分区：
文献类型：
--
作者：
Pisarev, AV;Kolupaeva, VG;Pestova, TV
真核起始因子（Eukaryotic initiation factor, eIF） 1维持了起始密码子选择的保真度，使哺乳动物43S预起始复合物能够区分偏离barG下最优序列GCC(a /G)CC （AUG）的AUG密码子，其中（-）3和（+）4位置的嘌呤是最重要的。我们假设eIF1的作用是对抗48S起始复合物形成过程中密码子-反密码子碱基配对时核糖体复合物中发生的构象变化，而（-）3和（+）4背景核苷酸的作用是通过与该复合物的组分相互作用来稳定这些变化。在这里，我们报道了U和G at(+)4在48S复合物中都与核糖体蛋白（rp） S15进行紫外交联。然而，在18S rRNA的第44螺旋上，U与C-1696交联强烈，与AA（1818-1819）交联较弱，而G只与AA（1818-1819）交联。U at(-)3与rpS5和eIF2 α交联，而G仅与eIF2 α交联。紫外交联实验和使用α -亚基缺陷eIF2对48S复合物形成的分析结果表明，eIF2 α与（-）3嘌呤的相互作用是43S复合物识别（-）3上下文位置的原因，并表明（+）4嘌呤/AA（1818-1819）相互作用可能是识别（+）4位置的原因。
Eukaryotic initiation factor (eIF) 1 maintains the fidelity of initiation codon selection and enables mammalian 43S preinitiation complexes to discriminate against AUG codons with a context that deviates from the optimum sequence GCC(A/G)CC (AUG) under barG, in which the purines at (-)3 and (+)4 positions are most important. We hypothesize that eIF1 acts by antagonizing conformational changes that occur in ribosomal complexes upon codon-anticodon base-pairing during 48S initiation complex formation, and that the role of (-)3 and (+)4 context nucleotides is to stabilize these changes by interacting with components of this complex. Here we report that U and G at (+)4 both UV-cross-linked to ribosomal protein (rp) S15 in 48S complexes. However, whereas U cross-linked strongly to C-1696 and less well to AA(1818-1819) in helix 44 of 18S rRNA, G cross-linked exclusively to AA(1818-1819). U at (-)3 cross-linked to rpS5 and eIF2 alpha, whereas G cross-linked only to eIF2 alpha. Results of UV cross-linking experiments and of assays of 48S complex formation done using alpha-subunit-deficient eIF2 indicate that eIF2 alpha's interaction with the (-)3 purine is responsible for recognition of the (-)3 context position by 43S complexes and suggest that the (+)4 purine/AA(1818-1819) interaction might be responsible for recognizing the (+)4 position.