Mechanisms of translational enhancer elements in plant mRNA.

翻译增强子是提高编码蛋白质翻译效率的信使核糖核酸基序。在本研究项目之前，我们在植物中发现了两个翻译增强子元件。该项目的初步计划是利用标准的重组DNA技术来阐明这些基序的增强机制。然而，在研究进行到一半时，我们发现这些基序的增强作用被UTRs和编码区的核苷酸序列极大地改变了。这表明这些基序的增强作用不是自主的，而是基于碱基配对的复杂的分子内相互作用或对其敏感。对于如此复杂和微妙的翻译法规，还没有适用的实验方法。传统的碱基替换、插入和/或缺失技术可能会导致mRNA分子发生意想不到的构象变化，导致不可预见的碱基相互作用。在此基础上，我们在1999年改变了这个项目的进程，建立了适用于这种复杂的翻译增强的实验系统。经过两年的试错，借助体外进化理论和聚合酶链式反应技术，成功地建立了划时代的系统。这个系统允许我们在任何给定的mRNA分子上找到翻译增强子基序。对目前已获得的增强子基序的比较分析揭示了它们的共同特征：根据5‘帽和3’聚(A)尾巴的存在与否，它们的位置、序列和强度都不同。

Translational enhancers are mRNA motifs that enhance translational efficiency of encoded proteins. In advance of this research project, we had found two translational enhancer elements in plants. The initial plan of this project was to elucidate the enhancing mechanism of these motifs by use of standard recombinant DNA technology. However, halfway the research period, we found that the enhancer effects of these motifs are greatly altered by nucleotide sequences in the UTRs well as the coding region. This suggests that the enhancing effects of these motifs are not autonomous, but based on or sensitive to the complexed intra-molecular interactions of base pairing. There has been no experimental approach that applicable to such a complexed and delicate translational regulation. Conventional base substitution, insertion, and/or deletion technique may cause unexpected conformational change of mRNA molecules, resulting in unforseen base-interactions. From these, we altered the course of this project in 1999 to establish the experimental system applicable to such a complexed translational enhancement. After two years of trial and error, we succeeded in establishing the epoch-making system, with the aid of in vitro evolution theory and PCR technology. This system allows us to find translational enhancer motifs on any given mRNA molecules. Comparative analyses of the enhancer motifs thus far obtained revealed their common characteristics ; position, sequence and strength of then are different according to the presence or absence of 5' cap and 3' poly(A) tail.