Mechanisms of RNA binding and remodeling proteins

描述(申请人提供)：RNA结合和重塑蛋白质的机制RNA的抽象操作需要RNA结合蛋白和依赖于ATP的分子马达蛋白的作用，这些蛋白质被认为运输、重塑和解开RNA序列中的二级结构。这些分子马达中的许多都是死盒或密切相关的蛋白质。丙型肝炎病毒(丙型肝炎病毒)等正链RNA病毒需要这些蛋白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从总体上理解RNA的新陈代谢是至关重要的，并将促进我们对RNA病毒是如何复制的理解。该项目的目标是通过丙型肝炎病毒的RNA结合蛋白和RNA重塑酶来确定RNA重塑的机制。非结构蛋白3(NS3)是一种RNA重塑酶(或解旋酶)，是丙型肝炎病毒复制所必需的。我们提出了一种新的RNA重塑模型，通过这种酶，寡聚体酶的多个亚基共同作用，融化RNA中的二级结构。我们的模型预测，NS3以缓慢的动力学步骤参与双链的缓慢解旋，随后以依赖于ATP的方式快速易位。我们将通过生物物理、生化和生物实验的组合来测试这个模型。我们将使用蛋白质足迹和质谱学相结合的方法来确定蛋白质相互作用的特定位置。不形成寡聚体结构的截短形式的NS3将被检查，以确定蛋白质-蛋白质相互作用在RNA重塑中所起的具体作用。我们的工作已经确定NS5A是一种与自身和NS3相互作用的RNA结合蛋白。NS5A的结构代表了核酸识别的新折叠，我们准备揭示该蛋白质与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阻碍二聚化的NS5A变体，然后测试这些变体的RNA结合活性和支持细胞中的丙型肝炎病毒复制来确定。将使用新的生化和生物学方法详细研究NS3和NS5A之间的相互作用。在目标1中，我们将测试我们的假设，通过NS3进行RNA重塑。在目标2中，我们将确定NS5A的二聚化在RNA结合中的作用。在目标3中，我们将检验NS5A作为NS3解旋酶活性的加工性因子的假设。确定丙型肝炎病毒RNA结合和重塑蛋白的机制将揭示新的分子方法来破坏导致丙型肝炎病毒复制的途径。因此，了解蛋白质结合和操纵RNA的分子机制具有重要的生物学和医学意义。 公共卫生相关性：RNA结合的机制和重塑蛋白质叙述：结合和操纵RNA的蛋白质对许多生物学过程至关重要，包括翻译、转录和基因调控。RNA结合蛋白在丙型肝炎病毒等病毒中也发挥着关键作用，丙型肝炎病毒感染着世界上近3%的人口。超过75%的丙型肝炎病毒感染无法痊愈，导致持续的病毒感染，可能导致肝纤维化，并逐渐发展为严重和致命的疾病，包括肝硬变和肝癌。确定丙型肝炎病毒RNA结合和重塑蛋白的机制将揭示新的分子方法来破坏导致丙型肝炎病毒复制的途径。因此，了解蛋白质结合和操纵RNA的分子机制具有广泛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.