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。由RNA激活的干扰素诱导的蛋白激酶（PKR）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。在长链双链RNA（dsRNA）（通常是病毒来源的）存在下，PKR通过自磷酸化而被激活。一旦被激活，磷酸化的PKR可以磷酸化真核起始因子-2 α（eIF-2 α），导致翻译起始的抑制，在某些情况下，导致程序性细胞死亡或凋亡。 PKR也被证明是人类免疫缺陷病毒1型（HIV-1）复制的调节因子，并被认为是肿瘤抑制因子。因此，PKR作用的机制对于人类健康相关研究的许多不同领域具有中心兴趣和重要性。 不幸的是，PKR激活的详细机制知之甚少。 本研究计划的重点是阐明PKR激活和RNA调控的动力学机制。将建立PKR在与dsRNA的非序列特异性相互作用后组装成活化复合物的详细动力学框架。 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的内源荧光的停流研究、平衡荧光结合研究和定点诱变。PKR也可以由含有专门的非dsRNA或非沃森-克里克结构的病毒和细胞RNA调节。详细的机制和结构的几个非沃森克里克RNA能够调节PKR将被检查。上述机制实验的结果将有助于发展一个动力学框架，在该框架内分配和理解结构化RNA的不同作用。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结构也将通过包括结构映射、交联、足迹法和几种新型体外选择方法的方法进行检查。将选择和富集对调节PKR功能至关重要的专门RNA，目的是确定一组规则，这些规则将允许预测病毒或细胞RNA是PKR的正调节剂还是负调节剂。