Karginov A V, Lodder M, Hecht S M
Department of Chemistry and Department of Biology, University of Virginia, Charlottesville, VA 22901, USA.
Nucleic Acids Res. 1999 Aug 15;27(16):3283-90. doi: 10.1093/nar/27.16.3283.
A new strategy for studying the mechanism of translation initiation in eukaryotes has been developed. The strategy involves the use of an in vitro translation system to incorporate a non-natural fluorescent amino acid into a protein from a suppressor tRNAPheCUA misacylated with that amino acid. It is thereby possible to monitor translation initiation efficiency at an AUG codon in different contexts; this is illustrated for three constructs encoding Escherichia coli dihydrofolate reductase mRNA with different translation initiation regions. Fluorescence measurements after in vitro translation of the mRNAs in rabbit reticulocyte lysate reflected differences in the position and efficiency of translation initiation and, therefore, can be used for characterization of the translation initiation process.
一种研究真核生物翻译起始机制的新策略已经被开发出来。该策略涉及使用体外翻译系统，将一种非天然荧光氨基酸掺入到由错配酰化该氨基酸的抑制性tRNAPheCUA所合成的蛋白质中。由此可以监测不同背景下AUG密码子处的翻译起始效率；这在三种编码具有不同翻译起始区域的大肠杆菌二氢叶酸还原酶mRNA的构建体中得到了说明。在兔网织红细胞裂解物中对mRNA进行体外翻译后的荧光测量反映了翻译起始位置和效率的差异，因此可用于表征翻译起始过程。
