Selection of initiation sites by eucaryotic ribosomes: effect of inserting AUG triplets upstream from the coding sequence for preproinsulin.
Selection of initiation sites by eucaryotic ribosomes: effect of inserting AUG triplets upstream from the coding sequence for preproinsulin.
复制标题
真核核糖体起始位点的选择：在前胰岛素原编码序列上游插入 AUG 三联体的影响。
作者：
M. Kozak
在SV 40早期启动子的指导下直接合成大鼠前胰岛素原的重组质粒已被用于探测翻译起始的机制。插入上游AUG三联体，其相对于前胰岛素原的编码序列是框外的，降低了胰岛素原的产量，与扫描模型的预测一致。上游AUG密码子干扰的程度取决于AUG三联体周围的序列;对于两种构建体（p255 /20和C2），5 '近端AUG密码子构成了绝对屏障：前胰岛素原的下游起始位点没有起始。然而，用另外两个构建体（p255 /9，p255 /21），尽管在有利的起始环境中存在上游的框外AUG密码子，仍产生胰岛素原。在这些情况下，由第一个AUG三联体设定的阅读框架很短，在前胰岛素原编码序列开始之前终止。核糖体起始于第一个AUG，终止，然后再起始于前胰岛素原编码序列之前的AUG的解释是通过引入消除终止密码子的点突变来测试的：所产生的突变体不产生胰岛素原。
Recombinant plasmids that direct synthesis of rat preproinsulin under the direction of the SV40 early promoter have been used to probe the mechanism of initiation of translation. Insertion of an upstream AUG triplet that was out-of-frame with respect to the coding sequence for preproinsulin reduced the yield of proinsulin, in keeping with the predictions of the scanning model. The extent to which an upstream AUG codon interfered depended on sequences surrounding the AUG triplet; with two constructs ( p255 /20 and C2) the 5'-proximal AUG codon constituted an absolute barrier: there was no initiation at the downstream start site for preproinsulin. With two other constructs ( p255 /9, p255 /21), however, proinsulin was made despite the presence of an upstream, out-of-frame AUG codon in a favorable context for initiation. In those cases the reading frame set by the first AUG triplet was short, terminating before the start of the preproinsulin coding sequence. The interpretation that ribosomes initiate at the first AUG, terminate, and then reinitiate at the AUG that directly precedes the preproinsulin coding sequence was tested by introducing a point mutation that eliminated the terminator codon: the resulting mutant made no proinsulin.