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

描述（由申请人提供）：DEAD盒蛋白是超家族2 RNA解旋酶，其是结构化RNA进行的几乎每一个过程所需的，从前mRNA剪接和翻译到蛋白质和RNA的细胞内运输。它们也是复制包括HIV-1和HCV在内的病毒所必需的，并且过度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。从机制研究的见解将是至关重要的基本生物过程的深入理解和理解，并最终治疗重要的病毒性疾病和癌症。我们集中在真菌死亡盒蛋白CYT-19和Mss 116 p，其功能作为一般的RNA伴侣在线粒体组I和组II自我剪接内含子的折叠。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，并且它们的催化活性为天然状态的形成提供了稳健和灵敏的读数。在第1-5年的资助（2004-2009年），我们使用了一个充分研究的组I内含子从嗜热四膜虫显示，CYT-19的功能作为一个真正的伴侣，促进长寿命的错误折叠构象的重折叠，而不起作用的下游催化步骤。我们发现CYT-19非特异性地破坏结构，不区分天然构象和错误折叠构象，并且这种活性有利于天然RNA的积累，因为它比错误折叠构象稳定得多。自从提交了获得两年ARRA资助（2009-2011）的续约申请以来，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可以强烈抑制三级结构，解开一个短螺旋后，螺旋自发'undocks'与内含子核心的三级接触。快速动力学实验也支持这样的模型，其中CYT-19的C-末端“尾”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依赖性构象变化的单个循环中完全解旋短RNA螺旋。总之，这项工作导致了一个通用模型的RNA分子伴侣活性的死亡盒蛋白质，其中蛋白质被定位到结构化的RNA通过拴系，他们破坏暴露的二级结构的元素非proc，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测试和扩展这个模型。在目标1和2中，我们将使用简单的双链底物和实验方法来探测ATP酶循环如何与RNA解旋耦合，以及在多大程度上束缚限制解旋酶核心的位置和方向。在目标3和4中，我们将使用物理和化学方法来跟踪依赖于CYT-19和Mss 116 p的I组和II组内含子在体内的折叠，测试特定的假设并探索我们的一般分子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.