Mechanisms of RNA binding and remodeling proteins

描述（申请人提供）：RNA结合和重塑蛋白的机制摘要RNA的操纵需要RNA结合蛋白和ATP依赖性分子马达蛋白的作用，所述分子马达蛋白被认为在RNA序列中运输、重塑和展开二级结构。许多这些分子马达是死亡盒或密切相关的蛋白质。正链RNA病毒如丙型肝炎病毒（HCV）需要这些蛋白质的活性来进行病毒复制。确定这些蛋白质的分子机制对于我们理解RNA代谢具有根本的重要性，并将促进我们对RNA病毒如何复制的理解。该项目的目标是确定RNA结合蛋白和RNA重塑酶从丙型肝炎病毒的RNA重塑的机制。非结构蛋白3（NS 3）是一种RNA重塑酶（或解旋酶），是HCV复制所必需的。我们提出了一个新的模型，RNA重塑这种酶，即多个亚基的寡聚酶一起工作，融化出二级结构的RNA。我们的模型预测，NS 3参与在缓慢的动力学步骤的双链体的缓慢解旋，然后以ATP依赖的方式快速易位。我们将使用生物物理学、生物化学和生物学实验的组合来测试这个模型。我们将使用蛋白质足迹结合质谱法来确定蛋白质-蛋白质相互作用的特定位点。将检查不形成寡聚体结构的NS 3的截短形式以确定蛋白质-蛋白质相互作用在RNA重塑中发挥的特定作用。我们的工作已经确定NS 5A是一种RNA结合蛋白，它与自身和NS 3相互作用。NS 5A的结构代表了核酸识别的新折叠，我们准备揭示这种蛋白质与RNA结合的结构/功能关系。蛋白质-蛋白质相互作用的重要性将通过制备二聚化受阻的NS 5A变体，然后测试这些变体的RNA结合活性和对细胞中HCV复制的支持来确定。NS 3和NS 5A之间的相互作用将使用新的生物化学和生物学方法进行详细研究。在目标1中，我们将通过NS 3来验证我们对RNA重塑的假设。在目标2中，我们将确定NS 5A二聚化在RNA结合中的作用。在目标3中，我们将检验NS 5A作为NS 3解旋酶活性的持续合成因子的假设。确定HCV RNA结合和重塑蛋白的机制将揭示新的分子方法来破坏负责HCV复制的途径。因此，了解结合和操纵RNA的蛋白质的分子机制具有生物学和医学意义。 公共卫生相关性：RNA结合和重塑蛋白质的机制叙述：结合和操纵RNA的蛋白质对许多生物过程（包括翻译，转录和基因调控）具有根本的重要性。RNA结合蛋白在病毒中也起着关键作用，例如丙型肝炎病毒，其感染了世界上近3%的人口。超过75%的HCV感染从未消退，导致持续的病毒感染，可导致肝纤维化，并逐渐导致严重和致命的疾病，包括肝硬化和肝癌。确定HCV RNA结合和重塑蛋白的机制将揭示新的分子方法来破坏负责HCV复制的途径。因此，了解结合和操纵RNA的蛋白质的分子机制具有普遍的生物学和医学意义。

DESCRIPTION (provided by applicant): Mechanisms of RNA binding and remodeling proteins Abstract Manipulation of RNA requires the action of RNA binding proteins and ATP-dependent, molecular motor proteins that are believed to transport, remodel, and unwind secondary structures in RNA sequences. Many of these molecular motors are DEAD-box or closely related proteins. Positive strand RNA viruses such as the Hepatitis C virus (HCV) require the activity of these proteins for viral replication. Determination of the molecular mechanisms of these proteins is of fundamental importance to our understanding of RNA metabolism in general and will advance our understanding of how RNA virus' are replicated. The goal of this project is to determine the mechanism of RNA remodeling by an RNA binding protein and an RNA remodeling enzyme from the Hepatitis C virus. Non-structural protein 3 (NS3) is an RNA remodeling enzyme (or helicase), that is necessary for HCV replication. We propose a new model for RNA remodeling by this enzyme whereby multiple subunits of an oligomeric enzyme work together to melt out secondary structures in RNA. Our model predicts that NS3 participates in slow untwisting of the duplex in slow kinetic step, followed by rapid translocation in an ATP-dependent manner. We will test this model by using a combination of biophysical, biochemical, and biological experiments. We will identify the specific sites of protein-protein interactions by using protein footprinting coupled with mass spectrometry. Truncated forms of NS3 that do not form oligomeric structures will be examined to determine the specific roles that protein-protein interactions play in RNA remodeling. Our work has identified NS5A as an RNA binding protein that interacts with itself and with NS3. The structure of NS5A represents a new fold in nucleic acid recognition, and we are poised to uncover the structure/function relationship for RNA binding by this protein. The importance of protein-protein interactions will be determined by preparing variants of NS5A that are impeded in dimerization, followed by testing of those variants for RNA binding activity and for support of HCV replication in cells. The interplay between the NS3 and NS5A will be examined in detail using new biochemical and biological approaches. In aim 1, we will test our hypothesis for RNA remodeling by NS3. In aim 2, we will determine the role of dimerization of NS5A in RNA binding. In aim 3, the we will test the hypothesis that NS5A serves as a processivity factor for NS3 helicase activity. Determining the mechanisms of HCV RNA binding and remodeling proteins will reveal new molecular methods to disrupt the pathways responsible for HCV replication. Therefore, understanding the molecular mechanisms of proteins that bind and manipulate RNA is of biological and medical significance. PUBLIC HEALTH RELEVANCE: Mechanisms of RNA binding and remodeling proteins Narrative: Proteins that bind and manipulate RNA are of fundamental importance to many biological processes including translation, transcription, and gene regulation. RNA binding protiens also play key roles in viruses such as the Hepatitis C Virus, which infects almost 3 % of the world's population. Over 75% of HCV infections never resolve, resulting in persistent virus infection that can lead to liver fibrosis and, progressively, to severe and fatal diseases, including liver cirrhosis and liver cancer. Determining the mechanisms of HCV RNA binding and remodeling proteins will reveal new molecular methods to disrupt the pathways responsible for HCV replication. Therefore, understanding the molecular mechanisms of proteins that bind and manipulate RNA is of general biological and medical significance.