Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.
EMBO J. 2010 Nov 17;29(22):3819-25. doi: 10.1038/emboj.2010.255. Epub 2010 Oct 12.
Bacterial ribosomes stalled by truncated mRNAs are rescued by transfer-messenger RNA (tmRNA), a dual-function molecule that contains a tRNA-like domain (TLD) and an internal open reading frame (ORF). Occupying the empty A site with its TLD, the tmRNA enters the ribosome with the help of elongation factor Tu and a protein factor called small protein B (SmpB), and switches the translation to its own ORF. In this study, using cryo-electron microscopy, we obtained the first structure of an in vivo-formed complex containing ribosome and the tmRNA at the point where the TLD is accommodated into the ribosomal P site. We show that tmRNA maintains a stable 'arc and fork' structure on the ribosome when its TLD moves to the ribosomal P site and translation resumes on its ORF. Based on the density map, we built an atomic model, which suggests that SmpB interacts with the five nucleotides immediately upstream of the resume codon, thereby determining the correct selection of the reading frame on the ORF of tmRNA.
细菌核糖体被截断的 mRNA 所阻遏，可被转移信使 RNA（tmRNA）拯救，后者是一种具有 tRNA 样结构域（TLD）和内部开放阅读框（ORF）的双功能分子。tmRNA 用其 TLD 占据空的 A 位，在延伸因子 Tu 和一种称为小蛋白 B（SmpB）的蛋白因子的帮助下进入核糖体，并将翻译切换到其自身的 ORF。在这项研究中，我们使用冷冻电子显微镜获得了第一个包含核糖体和 tmRNA 的活体形成的复合物的结构，此时 TLD 被容纳到核糖体的 P 位。我们表明，当 tmRNA 的 TLD 移动到核糖体的 P 位并在其 ORF 上重新开始翻译时，tmRNA 在核糖体上保持稳定的“弧叉”结构。基于密度图，我们构建了一个原子模型，该模型表明 SmpB 与重新开始密码子上游的五个核苷酸相互作用，从而确定了 tmRNA 的 ORF 上正确的阅读框选择。
