MECHANISM FOR REGULATION OF PKR PROTEIN BY RNA

应对和阻止病毒复制的细胞机制对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)介导了人类病毒的防御机制，以及人类细胞的生长、分化和程序性死亡。在存在典型的病毒来源的长片段双链RNA(DsRNA)的情况下，PKR通过自身磷酸化而激活。一旦被激活，磷酸化的PKR就可以磷酸化真核细胞启动因子-2α(eIF-2α)，导致翻译的启动被抑制，在某些情况下，细胞程序性死亡或凋亡。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，并被认为是一种肿瘤抑制因子。因此，PKR的作用机制对人类健康相关研究的许多不同领域具有中心意义和重要性。遗憾的是，PKR激活的详细机制尚不清楚。本研究的重点是阐明PKR激活的动力学机制和RNA对其的调控。将建立一个详细的动力学框架，用于在与dsRNA非序列特异性相互作用时将PKR组装成激活的复合体。这将通过机械酶学和生物化学的方法实现，包括利用PKR或标记RNA的固有荧光的停流研究、平衡荧光结合研究和定点突变。PKR也可以受病毒和细胞RNA的调控，这些RNA含有特殊的非dsRNA或非Watson-Crick结构。我们将研究几种能够调节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开发一个动力学框架，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结构也将通过结构映射、交联、足迹和几种新的体外选择方法进行检测。将选择和丰富对调节PKR功能至关重要的专门RNA，目的是确定一套规则，允许预测病毒或细胞RNA是PKR的正调控因子还是负调控因子。

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.