Meng S Y, Hui J O, Haniu M, Tsai L B
Department of Microbiology and Applied Microbial Genetics, Amgen Inc., Thousand Oaks, CA 91320-1789, USA.
Biochem Biophys Res Commun. 1995 Jun 6;211(1):40-8. doi: 10.1006/bbrc.1995.1775.
A highly efficient UGA stop codon readthrough event during the synthesis of human neurotrophin 3 in E. coli is described. The incorporation of a Trp residue at the UGA stop codon is confirmed combining both the chemical analyses and the molecular and genetic data in this report. The 3' adjacent nuleotide to the UGA stop codon plays a crucial role in determining the readthrough efficiency in the order of A > G > C > U. The replacement of UGA with UAA or UAG totally abolished this readthrough phenomenon and the use of StpR host cells also prevented the occurrence of UGA readthrough. Gene dosage (or plasmid copy number) effect was not indicated in this event; however, the titration of RF-2 by mRNA transcripts under over-expression conditions might explain why tRNAtrp competes so well with RF-2 for UGA. Another apparently less produced readthrough product resulting from a transcript with no stop codon is also recorded, and the addition of a second in-frame stop codon increased the amount of the observed readthrough product.
本文描述了在大肠杆菌中合成人神经营养因子3期间发生的高效UGA终止密码子通读事件。结合本报告中的化学分析以及分子和遗传数据，证实了在UGA终止密码子处掺入了一个色氨酸残基。UGA终止密码子的3'相邻核苷酸在决定通读效率方面起着关键作用，顺序为A > G > C > U。用UAA或UAG取代UGA完全消除了这种通读现象，并且使用StpR宿主细胞也阻止了UGA通读的发生。此事件中未显示基因剂量（或质粒拷贝数）效应；然而，在过表达条件下mRNA转录本对RF-2的滴定可能解释了为什么tRNAtrp与RF-2在UGA处竞争得如此激烈。还记录了另一种显然产量较低的由无终止密码子的转录本产生的通读产物，并且添加第二个读框内终止密码子增加了观察到的通读产物的量。
