Tessier L H, Sondermeyer P, Faure T, Dreyer D, Benavente A, Villeval D, Courtney M, Lecocq J P
Nucleic Acids Res. 1984 Oct 25;12(20):7663-75. doi: 10.1093/nar/12.20.7663.
Parameters influencing the efficiency of expression of the human immune interferon (IFN-gamma) gene in E. coli were studied by comparing a series of eight in vitro-derived gene variants. These contained all possible combinations of silent mutations in the first three codons of the mature IFN-gamma polypeptide coding sequence. Expression levels varied up to 50-fold among the different constructions. Comparison of messenger RNA secondary structure models for each variant suggested that the presence of stem-loop structures blocking the translation initiation signals could drastically decrease the efficiency of IFN-gamma synthesis. With variants displaying no stable mRNA secondary structure in the region, a C----U transition at position +11 after the AUG resulted in a 5-fold increase in expression indicating that RNA primary structure also plays an important role in expression. In addition we demonstrate that, in this system, a spacing of 8 nucleotides between the Shine-Dalgarno region and AUG was optimal for gene expression and that the steady-state production level of IFN-gamma rose exponentially with increasing rate of synthesis.
通过比较一系列八个体外衍生的基因变体，研究了影响人免疫干扰素（IFN-γ）基因在大肠杆菌中表达效率的参数。这些变体包含成熟IFN-γ多肽编码序列前三个密码子中所有可能的沉默突变组合。不同构建体之间的表达水平差异高达50倍。对每个变体的信使RNA二级结构模型进行比较表明，存在阻碍翻译起始信号的茎环结构会大幅降低IFN-γ合成的效率。对于该区域中未显示稳定mRNA二级结构的变体，AUG后第11位的C→U转换导致表达增加5倍，这表明RNA一级结构在表达中也起着重要作用。此外，我们证明，在该系统中，Shine-Dalgarno区域与AUG之间8个核苷酸的间隔对于基因表达是最佳的，并且IFN-γ的稳态产生水平随着合成速率的增加呈指数上升。
