Lainé Sébastien, Thouard Anne, Komar Anton A, Rossignol Jean-Michel
Laboratoire de Génétique et Biologie Cellulaire, UMR 81 59 CNRS/Université Versailles St-Quentin/EPHE, France.
Gene. 2008 Apr 15;412(1-2):95-101. doi: 10.1016/j.gene.2008.01.018. Epub 2008 Feb 7.
In Escherichia coli the rare codons AGG, AGA and CGA are reported to have a detrimental effect on protein synthesis, especially during the expression of heterologous proteins. In the present work, we have studied the impact of successive clusters of these rare codons on the accuracy of mRNA translation in E. coli. For this purpose, we have analyzed the expression of an mRNA which contains in its 3' region a triplet and a tandem of AGA codons. This mRNA is derived from the human hepatitis B virus (HBV) preC gene. Both in eukaryotic cells and in eukaryotic cell-free translation system, this mRNA, directs the synthesis of a single 25 kDa protein. However, in a conventional E. coli strain BL 21 (DE3), transformed with a plasmid expressing this protein the synthesis of four polypeptides ranging from 30 to 21.5 kDa can be observed. Using different approaches, notably expression of i) precore mutated proteins or ii) chimeric proteins containing HA- and Myc-tags downstream of the AGA clusters (respectively in the -1 or +1 frame), we have found that when the ribosome encounters the AGA clusters, it can then resume the translation in both +1 and -1 frames. This result is in agreement with the model proposed recently by Baranov et al. (Baranov, P.V., Gesteland, R.F., Atkins, J.F., 2004. P-site tRNA is a crucial initiator of ribosomal frameshifting. RNA 10, 221-230), thus confirming that AGA/AGG codons can serve as sites for -1 frameshifting events. Only +1 frameshifting was suggested previously to occur at the AGA/AGG clusters.
据报道，在大肠杆菌中，稀有密码子AGG、AGA和CGA对蛋白质合成具有有害影响，尤其是在异源蛋白质表达过程中。在本研究中，我们研究了这些稀有密码子的连续簇对大肠杆菌中mRNA翻译准确性的影响。为此，我们分析了一种mRNA的表达，该mRNA在其3'区域包含一个三联体和一个AGA密码子串联。这种mRNA来源于人类乙型肝炎病毒（HBV）前C基因。在真核细胞和真核无细胞翻译系统中，这种mRNA都指导合成一种单一的25 kDa蛋白质。然而，在用表达该蛋白质的质粒转化的传统大肠杆菌菌株BL 21（DE3）中，可以观察到合成了四种分子量在30至21.5 kDa之间的多肽。使用不同的方法，特别是i）前核心突变蛋白的表达或ii）在AGA簇下游（分别在-1或+1框架中）包含HA-和Myc-标签的嵌合蛋白的表达，我们发现当核糖体遇到AGA簇时，它可以在+1和-1框架中恢复翻译。这一结果与Baranov等人最近提出的模型一致（Baranov, P.V., Gesteland, R.F., Atkins, J.F., 2004. P-site tRNA是核糖体移码的关键起始因子。RNA 10, 221-230），从而证实AGA/AGG密码子可作为-1移码事件的位点。此前仅有人提出在AGA/AGG簇处会发生+1移码。
