Sørensen M A, Kurland C G, Pedersen S
Institute of Microbiology, University of Copenhagen, Denmark.
J Mol Biol. 1989 May 20;207(2):365-77. doi: 10.1016/0022-2836(89)90260-x.
We wish to determine whether differences in translation rate are correlated with differences in codon usage or with differences in mRNA secondary structure. We therefore inserted a small DNA fragment in the lacZ gene either directly or flanked by a few frame-shifting bases, leaving the reading frame of the lacZ gene unchanged. The fragment was chosen to have "infrequent" codons in one reading frame and "common" codons in the other. The insert in these constructs does not seem to give mRNAs that are able to form extensive secondary structures. The translation time for these modified lacZ mRNAs was measured with a reproducibility better than plus or minus one second. We found that the mRNA with infrequent codons inserted has an approximately three-seconds longer translation time than the one with common codons. In another set of experiments we constructed two almost identical lacZ genes in which the lacZ mRNAs have the potential to generate stem structures with stabilities of about -75 kcal/mol. In this way we could investigate the influence of mRNA structure on translation rate. This type of modified gene was generated in two reading frames with either common or infrequent codons similar to our first experiments. We find that the yield of protein from these mRNAs is reduced, probably due to the action in vivo of an RNase. Nevertheless, the data do not indicate that there is any effect of mRNA secondary structure on translation rate. In contrast, our data persuade us that there is a difference in translation rate between infrequent codons and common codons that is of the order of sixfold.
我们希望确定翻译速率的差异是否与密码子使用的差异或mRNA二级结构的差异相关。因此，我们在lacZ基因中直接插入一个小DNA片段，或者在其两侧插入一些移码碱基，而不改变lacZ基因的阅读框。该片段被设计为在一个阅读框中有“罕见”密码子，而在另一个阅读框中有“常见”密码子。这些构建体中的插入片段似乎不会产生能够形成广泛二级结构的mRNA。这些修饰后的lacZ mRNA的翻译时间测量的重现性优于正负一秒。我们发现，插入罕见密码子的mRNA的翻译时间比插入常见密码子的mRNA长约三秒。在另一组实验中，我们构建了两个几乎相同的lacZ基因，其中lacZ mRNA有潜力形成稳定性约为 -75千卡/摩尔的茎环结构。通过这种方式，我们可以研究mRNA结构对翻译速率的影响。这种类型的修饰基因在两个阅读框中产生，带有与我们的第一个实验类似的常见或罕见密码子。我们发现，这些mRNA的蛋白质产量降低，可能是由于体内一种核糖核酸酶的作用。然而，数据并未表明mRNA二级结构对翻译速率有任何影响。相反，我们的数据使我们相信，罕见密码子和常见密码子之间的翻译速率存在约六倍的差异。
