蛋白质编码基因中的同义突变显著影响翻译效率。我们合成了一对编码绿色荧光蛋白的基因，这些基因被160个同义突变分开，以研究影响翻译效率的关键因素。一个序列针对大肠杆菌(GFP(Eco))进行优化,另一个针对枯草芽孢杆菌(GFP(Bsu))进行优化。当这些基因在大肠杆菌中表达时，GFP（Eco）发出的荧光比GFP（Bsu）强12倍，证实GFP(Bsu)基因的次优性质。然后我们采用定向进化来改善GFP(Bsu)的表达。随机诱变和DNA改组用于产生突变文库，对其进行荧光筛选。鉴定了显示6倍荧光增强的变体，其在Gly-4的罕见密码子中含有单个突变(G10A)。然而,取代产生了另一种罕见的密码子，AGA,对于Arg,这表明这种改善是由罕见密码子以外的因素引起的。我们接下来将饱和诱变应用于Gly-4。最暗的变体含有Gly-4的GGG密码子(GFP(Bsu)-G)。考虑到突变的位置，我们假设起始密码子周围mRNA二级结构的不稳定改善了表达。然后，我们将GFP（Bsu）的5'-非翻译区（5'UTR）中的核苷酸三联体随机化，与Gly-4密码子互补。鉴定了显示6倍荧光增强的变体，表现出不稳定的二级结构。当这个5'UTR序列与GFP(Bsu)-G组合时，实现了22倍的荧光改善。总的来说，起始密码子周围mRNA二级结构的稳定性主要影响翻译效率。
Synonymous mutations in protein coding genes significantly impact translation efficiency. We synthesized a pair of genes encoding green fluorescent protein that were separated by 160 synonymous mutations to investigate key factors that affect translation efficiency. One sequence was optimized for Escherichia coli (GFP(Eco)) and the other for Bacillus subtilis (GFP(Bsu)). When the genes were expressed in E. coli, GFP(Eco) fluoresced 12-fold stronger than GFP(Bsu), confirming the suboptimal nature of the GFP(Bsu) gene. We then employed directed evolution to improve the expression of GFP(Bsu). Random mutagenesis and DNA shuffling was used to generate mutant libraries, which were screened for fluorescence. A variant showing 6-fold fluorescence enhancement was identified, which contained a single mutation (G10A) in a rare codon for Gly-4. However, the substitution generated another type of rare codon, AGA, for Arg, suggesting that the improvement was caused by a factor other than the rare codon. We next applied saturation mutagenesis to Gly-4. The darkest variant contained a GGG codon (GFP(Bsu)-G) for Gly-4. Taking the location of the mutation into account, we hypothesized that destabilization of the mRNA secondary structure around the initiation codon improved the expression. We then randomized the nucleotide triplet in 5\'-untranslated region (5\'UTR) of GFP(Bsu), which is complementary to the Gly-4 codon. A variant showing 6-fold fluorescence enhancement was identified, which exhibited a destabilized secondary structure. When this 5\'UTR sequence was combined with GFP(Bsu)-G, 22-fold fluorescent improvement was achieved. Collectively, the stability of the mRNA secondary structure around the initiation codon predominantly affected the translation efficiency.
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