Udagawa Tsuyoshi, Shimizu Yoshihiro, Ueda Takuya
Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, FSB401, 5-1-5 Kashiwanoha, Kashiwa-shi, Chiba 277-8562, Japan.
J Biol Chem. 2004 Mar 5;279(10):8539-46. doi: 10.1074/jbc.M308784200. Epub 2003 Dec 11.
In eubacteria, the dissociation of the 70 S ribosome into the 30 S and 50 S subunits is the essential first step for the translation initiation of canonical mRNAs that possess 5'-leader sequences. However, a number of leaderless mRNAs that start with the initiation codon have been identified in some eubacteria. These have been shown to be translated efficiently in vivo. Here we investigated the process by which leaderless mRNA translation is initiated by using a highly reconstituted cell-free translation system from Escherichia coli. We found that leaderless mRNAs bind preferentially to 70 S ribosomes and that the leaderless mRNA.70 S.fMet-tRNA complex can transit from the initiation to the elongation phase even in the absence of initiation factors (IFs). Moreover, leaderless mRNA translation proceeds more efficiently if the intact 70 S ribosome is involved compared with the 30 S subunit. Furthermore, excess amounts of IF3 inhibit leaderless mRNA translation, probably because it promotes the disassembly of the 70 S ribosome into subunits. Finally, excess amounts of fMet-tRNA facilitate the IF-independent translation of leaderless mRNA. These observations strongly suggest that leaderless mRNA translation is initiated by the assembled 70 S ribosome and thereby bypasses the dissociation process.
在真细菌中，70 S核糖体解离为30 S和50 S亚基是具有5'前导序列的典型mRNA翻译起始的关键第一步。然而，在一些真细菌中已鉴定出许多以起始密码子开始的无前导mRNA。这些已被证明在体内能有效翻译。在此，我们使用来自大肠杆菌的高度重组的无细胞翻译系统研究了无前导mRNA翻译起始的过程。我们发现无前导mRNA优先结合70 S核糖体，并且无前导mRNA·70 S·fMet-tRNA复合物即使在没有起始因子（IFs）的情况下也能从起始阶段过渡到延伸阶段。此外，与30 S亚基相比，如果涉及完整的70 S核糖体，无前导mRNA的翻译效率更高。此外，过量的IF3会抑制无前导mRNA的翻译，可能是因为它促进了70 S核糖体解聚为亚基。最后，过量的fMet-tRNA促进了无前导mRNA的不依赖IF的翻译。这些观察结果强烈表明，无前导mRNA的翻译是由组装好的70 S核糖体起始的，从而绕过了解离过程。
