HDV RNA Folding and PKR Protein Regulation

HDV RNA 折叠和 PKR 蛋白调节

• 批准号: 7099466
• 负责人: PHILIP C BEVILACQUA
• 金额: $ 22.18万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7099466
• 关键词: Herpesviridae; biological signal transduction; conformation; double stranded RNA; enzyme activity; enzyme complex; enzyme mechanism; fluorescence resonance energy transfer; fluorescence spectrometry; genetic transcription; genetic translation; intermolecular interaction; nucleic acid structure; phosphorylation; protein folding; protein kinase; ribozymes; site directed mutagenesis; stop flow technique; surface plasmon resonance; thermodynamics; translation factor; virus RNA; virus protein

DESCRIPTION (provided by applicant): RNA is central to a variety of biological processes including transcription, splicing, translation, gene expression, development, and cell division. It is therefore of interest to understand how RNA folds into the structures necessary to carry out these functions. It is also important to understand how the structures of native RNA and RNA folding intermediates regulate critical biological processes including activation of the interferon-induced anti-viral agent protein kinase PKR. This proposal involves studying RNA folding events that occur during transcription, and understanding their similarities and differences to refolding events that occur upon addition of divalent ions. The importance of transcriptional pausing with the authentic template and polymerase will be investigated. In addition, the ability of non-native, or alternative pairings, to inhibit and, in selected cases, stimulate the folding of the catalytic RNA from hepatitis delta virus (HDV) will be studied. The influence of external factors, such as increased ionic strength and hepatitis delta antigen protein (HDAg), on resolution of alternative pairings will be systematically investigated. The extent to which these RNA folding states can activate PKR will be investigated as well. A recently discovered novel small RNA motif that activates PKR will be studied mechanistically. These studies will be carried out with biochemical and biophysical techniques, including PKR activation and rapid-quench RNA cleavage kinetics; stopped-flow fluorescence and absorbance kinetics; thermodynamic measurements; and RNA structure mapping. It is anticipated that these results may impact upon several areas relevant to human health including understanding replication of HDV, which increases the virulence of hepatitis B virus (HBV) infections, and regulation of PKR protein, which mediates part of the human viral defense mechanism. In addition, these studies may help uncover new roles for the kinase in vivo. Results are expected to be of fundamental interest to the RNA folding community and may impact the understanding of RNA folding in other biologically relevant RNA and RNA-protein systems including catalytic RNAs and the ribosome.

描述（由申请人提供）： RNA 对于多种生物过程至关重要，包括转录、剪接、翻译、基因表达、发育和细胞分裂。因此，了解 RNA 如何折叠成执行这些功能所需的结构是很有意义的。了解天然 RNA 和 RNA 折叠中间体的结构如何调节关键的生物过程（包括干扰素诱导的抗病毒剂蛋白激酶 PKR 的激活）也很重要。该提案涉及研究转录过程中发生的 RNA 折叠事件，并了解它们与添加二价离子时发生的重折叠事件的异同。将研究真实模板和聚合酶转录暂停的重要性。此外，还将研究非天然或替代配对抑制丁型肝炎病毒（HDV）催化RNA折叠的能力，并在选定的情况下刺激其折叠的能力。将系统地研究外部因素（例如增加的离子强度和丁型肝炎抗原蛋白（HDAg））对替代配对解析的影响。这些 RNA 折叠状态激活 PKR 的程度也将得到研究。最近发现的一种新型小 RNA 基序可激活 PKR，我们将对其进行机制研究。这些研究将利用生物化学和生物物理技术进行，包括 PKR 激活和快速淬灭 RNA 裂解动力学；停流荧光和吸光度动力学；热力学测量；和RNA结构图谱。 预计这些结果可能会影响与人类健康相关的多个领域，包括了解 HDV 的复制（增加乙型肝炎病毒 (HBV) 感染的毒力）以及 PKR 蛋白的调节（介导部分人类病毒防御机制）。此外，这些研究可能有助于揭示激酶在体内的新作用。预计结果将引起 RNA 折叠界的根本兴趣，并可能影响对其他生物学相关 RNA 和 RNA-蛋白质系统（包括催化 RNA 和核糖体）中 RNA 折叠的理解。