Department of Chemistry, Wayne State University, Detroit, Michigan 48202, United States.
Biochemistry. 2012 May 1;51(17):3531-8. doi: 10.1021/bi201751k. Epub 2012 Apr 19.
Transfer messenger RNA (tmRNA) and small binding protein B (SmpB) are the main components of the trans-translation rescue machinery that releases stalled ribosomes from defective mRNAs. Little is known about how SmpB binding affects the conformation of the tRNA-like domain (TLD) of tmRNA. It has been previously hypothesized that the absence of a D stem in the TLD provides flexibility in the elbow region of tmRNA, which can be stabilized by its interaction with SmpB. Here, we have used Förster resonance energy transfer to characterize the global structure of the tRNA-like domain of tmRNA in the presence and absence of SmpB and as a function of Mg(2+) concentration. Our results show tight and specific binding of SmpB to tmRNA. Surprisingly, our data show that the global conformation and flexibility of tmRNA do not change upon SmpB binding. However, Mg(2+) ions induce an 11 Å compaction in the tmRNA structure, suggesting that the flexibility in the H2a stem may allow different conformations of tmRNA as the TLD and mRNA-like domain need to be positioned differently while moving through the ribosome.
转移信使 RNA（tmRNA）和小结合蛋白 B（SmpB）是转译救援机制的主要组成部分，该机制可从有缺陷的 mRNA 上释放停滞的核糖体。关于 SmpB 结合如何影响 tmRNA 的 tRNA 样结构域（TLD）的构象，目前知之甚少。先前的假设是，TLD 中不存在 D 茎为 tmRNA 的肘区提供了灵活性，其与 SmpB 的相互作用可以稳定该灵活性。在这里，我们使用Förster 共振能量转移来表征 tmRNA 的 tRNA 样结构域在存在和不存在 SmpB 以及作为 Mg（2+）浓度函数时的整体结构。我们的结果显示 SmpB 与 tmRNA 的紧密和特异性结合。令人惊讶的是，我们的数据表明，SmpB 结合不会改变 tmRNA 的整体构象和灵活性。然而，Mg（2+）离子诱导 tmRNA 结构中 11 Å 的压缩，表明 H2a 茎的灵活性可能允许 tmRNA 具有不同的构象，因为 TLD 和 mRNA 样结构域在通过核糖体时需要以不同的方式定位。
