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Mistranslational errors associated with the rare arginine codon CGG in Escherichia coli

机译：与大肠杆菌中稀有精氨酸密码子CGG相关的翻译错误

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摘要

In Escherichia coli, CGG is a rare arginine codon occurring at a frequency of 0.54% in all E coli mRNAs or 9.8% when an arginine residue is encoded for. When present in high numbers or in clusters in highly expressed recombinant mRNA, rare codons can cause expression problems compromising product yield and translational fidelity. The coding region for an N-terminally polyhistidine tagged p27 protease domain from Herpes Simplex Virus 2 (HSV-2) contains 11 of these rare arginine codons, with 3 occurring in tandem near the C-terminus of the protein. When expressed in E coli, the majority of the recombinant material produced had an apparent molecular mass of 31 kDa by SDS-PAGE gels or 3 kDa higher than predicted. Detailed biochemical analysis was performed on chemical and enzymatic digests of the protein and peptide fragments were characterized by Edman and MS/MS sequencing approaches. Two major species were isolated comprising +1 frameshift events at both the second and third CGG codons in the triplet cluster. Translation proceeded in the missense frame to the next termination codon. In addition, significant levels of glutamine misincorporating for arginine were discovered, suggesting second base misreading of CGG as CAG. Coexpression of the argX gene, which encodes the cognate tRNA for CGG codons, largely eliminated both the frameshift and misincorporation events, and increased expression levels of authentic product by up to 7-fold. We conclude that supplementation of the rare arginyl tRNACGG levels by coexpression of the argX gene can largely alleviate the CGG codon bias present in E coli, allowing for efficient and accurate translation of heterologous gene products. (C) 2002 Elsevier Science (USA). All rights reserved. [References: 21]
机译：在大肠杆菌中，CGG是一种稀有的精氨酸密码子，在所有大肠杆菌mRNA中的出现频率为0.54％，当编码为精氨酸残基时，该频率为9.8％。当大量存在或以簇形式存在于高表达的重组mRNA中时，稀有密码子会引起表达问题，从而损害产品的产量和翻译保真度。来自单纯疱疹病毒2（HSV-2）的N端多组氨酸标记的p27蛋白酶结构域的编码区包含11个稀有精氨酸密码子，其中3个串联出现在该蛋白的C末端附近。当在大肠杆菌中表达时，通过SDS-PAGE凝胶产生的大多数重组材料的表观分子量为31 kDa，或比预期高3 kDa。对蛋白质的化学和酶消化物进行了详细的生化分析，并通过Edman和MS / MS测序方法对肽片段进行了表征。分离出两个主要物种，其在三重态簇中的第二和第三CGG密码子处都包含+1移码事件。翻译在错义框中进行到下一个终止​​密码子。另外，发现大量水平的谷氨酰胺误掺入精氨酸，表明CGG作为CAG的第二碱基误读。编码CGG密码子同源tRNA的argX基因的共表达在很大程度上消除了移码和错掺事件，并使真实产物的表达水平提高了多达7倍。我们得出结论，通过argX基因的共表达来补充稀有精氨酰tRNACGG的水平可以大大缓解大肠杆菌中存在的CGG密码子偏倚，从而实现异源基因产物的有效和准确翻译。 （C）2002 Elsevier Science（美国）。版权所有。 [参考：21]

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