Johansen H, Schümperli D, Rosenberg M
Proc Natl Acad Sci U S A. 1984 Dec;81(24):7698-702. doi: 10.1073/pnas.81.24.7698.
We have utilized a recombinant vector system designed to study gene control elements by fusing them to the Escherichia coli galactokinase gene (galK) to examine the effects of gene expression that result from introducing changes into the 5' noncoding leader region of the galK transcription unit. We varied the length of the 5' leader region and found no effect on galK expression, provided that the galK initiation codon remained the first AUG in the transcription unit. Using synthetic linkers, we then inserted specific sequences, each containing a single AUG codon at a defined position within the leader region. We found that the AUG inserts had widely different effects on galK expression and that the sequences surrounding the inserted AUG codons determined the magnitude of these effects. In addition, we placed these upstream inserted AUG codons into each of the three possible translation reading frames so that translation occurring in these frames terminates prior to, within, or downstream of the galK initiation codon, respectively. Single-nucleotide frameshift mutations were also introduced into one of these constructs to shift upstream translation into the other two possible reading frames. Depending upon where upstream translation stopped relative to the galK initiation codon, we observed consistently different effects on galK expression. Our results show that an upstream AUG that interferes with downstream translation initiation exerts its greatest effect when it translates out-of-frame through the downstream initiator into the gene. If translation is stopped upstream of or within the initiator, an unexpectedly high level of expression from the downstream AUG is maintained.
我们利用了一种重组载体系统，该系统通过将基因控制元件与大肠杆菌半乳糖激酶基因（galK）融合来研究基因控制元件，以检验因对galK转录单元的5'非编码前导区进行改变而导致的基因表达效应。我们改变了5'前导区的长度，发现只要galK起始密码子仍是转录单元中的第一个AUG，就对galK表达没有影响。然后，我们使用合成接头在该前导区内的特定位置插入特定序列，每个序列都包含一个单一的AUG密码子。我们发现这些AUG插入对galK表达有广泛不同的影响，并且插入的AUG密码子周围的序列决定了这些影响的程度。此外，我们将这些上游插入的AUG密码子置于三种可能的翻译阅读框中的每一种中，以使在这些阅读框中发生的翻译分别在galK起始密码子之前、之内或下游终止。还将单核苷酸移码突变引入其中一个构建体，以使上游翻译移至其他两种可能的阅读框中。根据上游翻译相对于galK起始密码子停止的位置，我们始终观察到对galK表达有不同的影响。我们的结果表明，干扰下游翻译起始的上游AUG在其通过下游起始密码子移码进入基因进行翻译时发挥最大作用。如果翻译在起始密码子上游或之内停止，则下游AUG会维持意外高水平的表达。
