Kozak M
Proc Natl Acad Sci U S A. 1986 May;83(9):2850-4. doi: 10.1073/pnas.83.9.2850.
Oligonucleotides designed to create hairpin structures were inserted upstream from the ATG initiator codon in several plasmids that encode preproinsulin, and the effects on translation were monitored in COS cells transfected by the vectors. Creation of a hairpin (delta G = -30 kcal/mol) that directly involves the ATG triplet at the start of the preproinsulin coding sequence does not reduce the yield of proinsulin. However, a more stable stem-and-loop structure (delta G = -50 kcal/mol) reduces the proinsulin yield by 85-95%. The stable hairpin inhibits even when it occurs at the midpoint of the 5' untranslated sequence and thus involves neither the cap nor the ATG codon. Presumably the migrating 40S ribosomal subunit can melt moderately stable duplexes but stalls at structures (delta G = -50 kcal/mol) that resist unfolding. Other experiments argue against the idea that sequestering the 5'-proximal ATG codon in a hairpin structure might allow it to be skipped by ribosomes in favor of an exposed ATG triplet farther downstream: when the primary sequence around the first ATG triplet is favorable for initiation, no translation from a downstream site can be detected, irrespective of whether the first ATG codon is single-stranded or base-paired.
设计用于形成发夹结构的寡核苷酸被插入到几个编码胰岛素原的质粒中ATG起始密码子的上游，并在由这些载体转染的COS细胞中监测其对翻译的影响。在胰岛素原编码序列起始处直接涉及ATG三联体的发夹结构（ΔG = -30千卡/摩尔）的形成不会降低胰岛素原的产量。然而，更稳定的茎环结构（ΔG = -50千卡/摩尔）会使胰岛素原产量降低85 - 95%。即使稳定的发夹结构出现在5'非翻译序列的中点，因而既不涉及帽结构也不涉及ATG密码子，它也会抑制翻译。据推测，迁移的40S核糖体亚基能够解开适度稳定的双链体，但会在抵抗解折叠的结构（ΔG = -50千卡/摩尔）处停滞。其他实验反驳了这样一种观点，即把5'近端的ATG密码子封闭在发夹结构中可能会使核糖体跳过它而优先选择下游一个暴露的ATG三联体：当第一个ATG三联体周围的一级序列有利于起始时，无论第一个ATG密码子是单链的还是碱基配对的，都检测不到来自下游位点的翻译。
