Physical and functional probing of DEAD-box proteins as general RNA chaperones

描述（由申请人提供）：DEAD-box蛋白是超家族2 RNA解旋酶，从mrna前剪接和翻译到蛋白质和RNA的细胞内运输，几乎所有结构化RNA进行的过程都需要它。它们也是包括HIV-1和HCV在内的病毒复制所必需的，而过表达与结肠癌和前列腺癌有关。这些蛋白质利用ATP结合和水解产生的能量来促进RNA的构象变化和折叠转变，但我们对它们如何操纵RNA结构的分子知识有限。来自机制研究的见解对于深入理解基本生物学过程以及理解和最终治疗重要的病毒性疾病和癌症至关重要。我们重点研究了真菌DEAD-box蛋白CYT-19和Mss116p，它们在线粒体I族和II族自剪接内含子的折叠中起着一般RNA伴侣的作用。这些系统对于机制研究是强大的，因为rna相对简单和易于处理，它们的催化活性为天然状态的形成提供了一个强大而敏感的读数。在资助的第1-5年（2004-2009年），我们使用了来自嗜热四膜虫（Tetrahymena thermomila）的一个经过充分研究的I组内含子来证明CYT-19作为一个真正的伴侣，促进长寿命错误折叠构象的重新折叠，而不参与下游催化步骤。我们发现CYT-19非特异性地破坏结构，不区分天然和错误折叠的构象，这种活性有利于天然RNA的积累，因为它比错误折叠的构象更稳定。自提交续期申请并获得两年ARRA资助（2009-2011）以来，我们取得了进一步的进展。单分子荧光和快速动力学直接表明，CYT-19可以受到三级结构的强烈抑制，只有在螺旋与内含子核心的三级接触中自发“解坞”后，才能解除短螺旋的缠绕。快速动力学实验也支持一个模型，其中CYT-19的c端“尾巴”接触结构化RNA并束缚解旋酶核心以破坏附近的结构，并且一系列速率测量表明，CYT-19在ATP依赖的构象变化的单周期中完全解绕短RNA螺旋。总之，这项工作通过DEAD-box蛋白得出了RNA伴侣活性的一般模型，其中蛋白质通过拴链定位到结构化RNA上，它们非程序性地破坏二级结构的暴露元素，使这些片段有机会形成新的接触。在这里，我们建议在两个重要的方向上测试和扩展这个模型。在目标1和目标2中，我们将使用简单的双底物和一系列实验方法来探索atp酶循环如何与RNA解绕耦合，以及系缚在多大程度上限制了解旋酶核心的位置和方向。在目标3和目标4中，我们将使用物理和化学方法跟踪体内依赖于CYT-19和Mss116p的I组和II组内含子的折叠，测试特定假设并探索我们的一般伴侣活性模型是否描述了这些蛋白质对其同源rna折叠的影响。

DESCRIPTION (provided by applicant): DEAD-box proteins are superfamily 2 RNA helicases that are required for virtually every process carried out by structured RNAs, from pre-mRNA splicing and translation to intracellular trafficking of proteins and RNAs. They are also required for replication of viruses including HIV-1 and HCV and overexpression is linked to colon and prostate cancer. These proteins use energy from ATP binding and hydrolysis to facilitate RNA conformational changes and folding transitions, but we have limited molecular knowledge about how they manipulate RNA structure. Insights from mechanistic studies will be critical for deep understanding of fundamental biological processes and for understanding and ultimately treatment of important viral diseases and cancer. We have focused on the fungal DEAD-box proteins CYT-19 and Mss116p, which function as general RNA chaperones in folding of mitochondrial group I and group II self-splicing introns. These systems are powerful for mechanistic studies because the RNAs are relatively simple and tractable, and their catalytic activity provides a robust and sensitive readout for formation of the native state. In years 1-5 of funding (2004-2009), we used a well-studied group I intron from Tetrahymena thermophila to show that CYT-19 functions as a true chaperone, facilitating refolding of a long-lived misfolded conformation without functioning in the downstream catalytic steps. We found that CYT-19 disrupts structure non-specifically, without distinguishing native from misfolded conformations, and this activity favors accumulation of the native RNA because it is much more stable than the misfolded conformation. Since submitting a renewal that received two years of ARRA funding (2009-2011), we made further advances. Single molecule fluorescence and rapid kinetics showed directly that CYT-19 can be strongly inhibited by tertiary structure, unwinding a short helix only after the helix spontaneously 'undocks' from tertiary contacts with the intron core. Rapid kinetics experiments also supported a model in which a C-terminal 'tail' of CYT-19 contacts structured RNA and tethers the helicase core to disrupt nearby structure, and a series of rate measurements indicated that CYT-19 completely unwinds short RNA helices in a single cycle of ATP- dependent conformational changes. Together, the work leads to a general model for RNA chaperone activity by DEAD-box proteins in which the proteins are localized to structured RNAs by tethering, and they disrupt exposed elements of secondary structure non-processively to allow these segments an opportunity to form new contacts. Here we propose to test and extend this model in two important directions. In Aims 1 and 2, we will use simple duplex substrates and an arsenal of experimental approaches to probe how the ATPase cycle is coupled to RNA unwinding and to what extent tethering constrains the position and orientation of the helicase core. In Aims 3 and 4 we will use physical and chemical approaches to follow folding of group I and group II introns that rely on CYT-19 and Mss116p in vivo, testing specific hypotheses and probing whether our model for general chaperone activity describes the effects of these proteins on folding of their cognate RNAs.