Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA.
Nucleic Acids Res. 2012 Jul;40(13):6199-207. doi: 10.1093/nar/gks278. Epub 2012 Mar 28.
Eukaryotic translation initiation is a highly regulated process in protein synthesis. The principal translation initiation factor eIF4AI displays helicase activity, unwinding secondary structures in the mRNAs 5'-UTR. Single molecule fluorescence resonance energy transfer (sm-FRET) is applied here to directly observe and quantify the helicase activity of eIF4AI in the presence of the ancillary RNA-binding factor eIF4H. Results show that eIF4H can significantly enhance the helicase activity of eIF4AI by strongly binding both to loop structures within the RNA transcript as well as to eIF4AI. In the presence of ATP, the eIF4AI/eIF4H complex exhibits persistent rapid and repetitive cycles of unwinding and re-annealing. ATP titration assays suggest that this process consumes a single ATP molecule per cycle. In contrast, helicase unwinding activity does not occur in the presence of the non-hydrolysable analog ATP-γS. Based on our sm-FRET results, we propose an unwinding mechanism where eIF4AI/eIF4H can bind directly to loop structures to destabilize duplexes. Since eIF4AI is the prototypical example of a DEA(D/H)-box RNA helicase, it is highly likely that this unwinding mechanism is applicable to a myriad of DEAD-box helicases employed in RNA metabolism.
真核翻译起始是蛋白质合成中高度调控的过程。主要的翻译起始因子 eIF4AI 显示解旋酶活性，解开 mRNA 5'-UTR 中的二级结构。这里应用单分子荧光共振能量转移（sm-FRET）来直接观察和定量辅助 RNA 结合因子 eIF4H 存在时 eIF4AI 的解旋酶活性。结果表明，eIF4H 可以通过强烈结合 RNA 转录本中的环结构以及 eIF4AI 来显著增强 eIF4AI 的解旋酶活性。在 ATP 的存在下，eIF4AI/eIF4H 复合物表现出持续的快速和重复的解旋和重新退火循环。ATP 滴定实验表明，该过程每循环消耗一个 ATP 分子。相比之下，在非水解类似物 ATP-γS 的存在下，解旋酶解旋活性不会发生。基于我们的 sm-FRET 结果，我们提出了一种解旋机制，其中 eIF4AI/eIF4H 可以直接结合环结构来使双链不稳定。由于 eIF4AI 是 DEA(D/H)-box RNA 解旋酶的典型范例，因此这种解旋机制极有可能适用于 RNA 代谢中使用的众多 DEAD-box 解旋酶。
