Single-molecule analysis of Mss116-mediated group II intron folding.-猫眼课题宝

DOI：

10.1038/nature09422

发表时间：

2010-10-21

期刊：

Nature

影响因子：

64.8

通讯作者：

中科院分区：

综合性期刊1区

文献类型：

作者：

来源链接：

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4484588/?report=classic

DEAD盒解旋酶是一种保守的酶，参与RNA代谢的几乎所有方面，但其作用机制仍不清楚。在这里，我们研究了死亡盒蛋白Mss 116对其天然底物，第II组内含子ai 5 γ的机制。II组内含子是结构复杂的催化RNA，被认为与真核剪接体进化相关，并且是大RNA折叠的有趣范例。我们使用单分子荧光来监测Mss 116对最小活性构建体ai 5 γ-D135的折叠动力学的影响。数据显示，Mss 116刺激状态之间的动态采样沿着折叠途径，以前观察到的效果只有高Mg 2+浓度。此外，数据表明，Mss 116促进折叠通过离散的ATP-独立和ATP-依赖的步骤。我们建议，Mss 116刺激组II内含子折叠通过一个多步骤的过程，涉及静电稳定的早期中间体和ATP水解在自然状态组装的最后阶段。

DEAD-box helicases are conserved enzymes involved in nearly all aspects of RNA metabolism, but their mechanisms of action remain unclear. Here, we investigated the mechanism of the DEAD-box protein Mss116 on its natural substrate, the group II intron ai5γ. Group II introns are structurally complex catalytic RNAs considered evolutionarily related to the eukaryotic spliceosome, and an interesting paradigm for large RNA folding. We used single-molecule fluorescence to monitor the effect of Mss116 on folding dynamics of a minimal active construct, ai5γ–D135. The data show that Mss116 stimulates dynamic sampling between states along the folding pathway, an effect previously observed only with high Mg2+ concentrations. Furthermore, the data indicate that Mss116 promotes folding through discrete ATP-independent and ATP-dependent steps. We propose that Mss116 stimulates group II intron folding through a multi-step process that involves electrostatic stabilization of early intermediates and ATP hydrolysis during the final stages of native state assembly.