INFLUENCES OF MESSENGER-RNA SECONDARY STRUCTURE ON INITIATION BY EUKARYOTIC RIBOSOMES
INFLUENCES OF MESSENGER-RNA SECONDARY STRUCTURE ON INITIATION BY EUKARYOTIC RIBOSOMES
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DOI：
10.1073/pnas.83.9.2850
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发表时间：
1986-05-01
影响因子：
11.1
通讯作者：
KOZAK, M
中科院分区：
文献类型：
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作者：
KOZAK, M
将设计成发夹结构的寡核苷酸插入到编码胰岛素前原的质粒中，在ATG启动子的上游插入，并在转染载体的COS细胞中监测对翻译的影响。发夹的创作（. delta）。G = -30 kcal/mol)，直接涉及胰岛素原前编码序列开始的ATG三重体，不会降低胰岛素原的产量。然而，更稳定的茎环结构（. delta）。G = -50 kcal/mol)可使胰岛素原产量降低85-95%。稳定发夹抑制即使发生在5‘’非翻译序列的中点，因此既不涉及帽也不涉及ATG密码子。据推测，迁移的40S核糖体亚基可以融化中等稳定的双链，但在结构上停滞不前。G = -50千卡/摩尔)，不易展开。其他实验反对这样一种观点，即在发夹结构中隔离5”-近端ATG密码子可能允许核糖体跳过它，从而有利于更远的下游暴露的ATG三联体：当第一个ATG三联体周围的初级序列有利于起始时，无论第一个ATG密码子是单链还是碱基配对，都无法检测到来自下游位点的翻译。
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 down-stream: when the primary sequence around the first ATG triplet is favorable for initiation, no translation from a down-stream site can be detected, irrespective of whether the first ATG codon is single-stranded or base-paired.