MECHANISM FOR REGULATION OF PKR PROTEIN BY RNA

RNA 调节 PKR 蛋白的机制

• 批准号: 6343052
• 负责人: PHILIP C BEVILACQUA
• 金额: $ 20.77万
• 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 2003-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6343052
• 关键词: atomic force microscopy; biological signal transduction; chemical binding; chemical kinetics; circular dichroism; conformation; double stranded RNA; enzyme activity; enzyme complex; enzyme mechanism; fluorescence resonance energy transfer; fluorescence spectrometry; genetic translation; host organism interaction; intermolecular interaction; molecular assembly /self assembly; nucleic acid structure; phosphorylation; protein kinase; site directed mutagenesis; stoichiometry; stop flow technique; surface plasmon resonance; translation factor; virus RNA

Cellular mechanisms for responding to and stopping virus replication are of critical importance for controlling human diseases. The interferon- induced protein kinase activated by RNA (PKR) mediates the human viral defense mechanism, as well as the growth, differentiation, and programmed death of human cells. In the presence of long stretches of double-stranded RNA (dsRNA), typically of viral origin, PKR becomes activated by autophosphorylation. Once activated, phosphorylated PKR can then phosphorylate eukaryotic initiation factor-2alpha (eIF-2alpha), causing inhibition of the initiation of translation and, in some cases, programmed cell death, or apoptosis. PKR has also been shown to be a regulator of human immunodeficiency virus type 1 (HIV-1) replication, and has been implicated as a tumor suppressor. The mechanism of PKR action is thus of central interest and importance to many different fields of human health-related research. Unfortunately, the detailed mechanism of PKR activation is poorly understood. This research proposal focuses on elucidating the kinetic mechanism for PKR activation and regulation by RNA. A detailed kinetic framework for the assembly of PKR into an activated complex upon non-sequence specific interactions with dsRNA will be established. This will be achieved by methods of mechanistic enzymology and biochemistry, including stopped-flow studies utilizing the intrinsic fluorescence of PKR or of tagged RNAs, equilibrium fluorescence binding studies, and site-directed mutagenesis. PKR can also be regulated by viral and cellular RNAs containing specialized non-dsRNA, or non-Watson-Crick, structures. The detailed mechanisms and structures of several non-Watson Crick RNAs that are able to regulate PKR will be examined. Results from the above mechanistic experiments will facilitate development of a kinetic framework within which to assign and understand the varied actions of the structured RNAs. This will be achieved by stopped-flow experiments, equilibrium fluorescence studies, and RNA-protein structure-function analysis. RNA structure will also be examined by approaches including structure mapping, crosslinking, footprinting, and several novel in vitro selection approaches. Specialized RNAs critical to regulating PKR function will be selected and enriched, with the goal of determining a set of rules that will allow prediction of whether a viral or cellular RNA is a positive or negative regulator of PKR.

响应和阻止病毒复制的细胞机制对于控制人类疾病至关重要。 RNA (PKR) 激活的干扰素诱导蛋白激酶介导人类病毒防御机制以及人类细胞的生长、分化和程序性死亡。当长链双链 RNA (dsRNA)（通常是病毒来源）存在时，PKR 通过自磷酸化而被激活。一旦被激活，磷酸化的 PKR 就会磷酸化真核起始因子-2α (eIF-2α)，从而抑制翻译起始，并在某些情况下导致程序性细胞死亡或凋亡。 PKR 还被​​证明是人类免疫缺陷病毒 1 型 (HIV-1) 复制的调节因子，并且被认为是一种肿瘤抑制因子。因此，PKR 作用机制对于人类健康相关研究的许多不同领域具有核心意义和重要性。 不幸的是，人们对 PKR 激活的详细机制知之甚少。 本研究计划的重点是阐明 RNA 激活和调节 PKR 的动力学机制。将建立一个详细的动力学框架，用于在与 dsRNA 进行非序列特异性相互作用时将 PKR 组装成激活的复合物。 这将通过机械酶学和生物化学方法来实现，包括利用 PKR 或标记 RNA 固有荧光的停流研究、平衡荧光结合研究和定点诱变。 PKR 还可以受到含有特殊非 dsRNA 或非 Watson-Crick 结构的病毒和细胞 RNA 的调节。将检查几种能够调节 PKR 的非 Watson Crick RNA 的详细机制和结构。上述机制实验的结果将有助于动力学框架的开发，在该框架内分配和理解结构化 RNA 的各种作用。这将通过停流实验、平衡荧光研究和 RNA-蛋白质结构-功能分析来实现。 RNA 结构也将通过结构作图、交联、足迹和几种新颖的体外选择方法等方法进行检查。将选择和丰富对调节 PKR 功能至关重要的专门 RNA，目的是确定一组规则，以预测病毒或细胞 RNA 是 PKR 的正调节剂还是负调节剂。