MECHANISM FOR REGULATION OF PKR PROTEIN BY RNA

响应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）存在的情况下，PKR被自磷酸化激活，通常是病毒起源。一旦被激活，磷酸化的PKR可以磷酸化真核起始因子-2alpha (eIF-2alpha)，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，并被认为是一种肿瘤抑制因子。因此，PKR的作用机制是人类健康相关研究许多不同领域的核心兴趣和重要性。不幸的是，PKR激活的详细机制尚不清楚。本研究拟重点阐明RNA激活和调控PKR的动力学机制。将建立PKR在与dsRNA非序列特异性相互作用时组装成活化复合体的详细动力学框架。这将通过机械酶学和生物化学的方法来实现，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诱变。PKR也可以被含有特殊非dsrna或非沃森-克里克结构的病毒和细胞rna调节。我们将研究几种能够调控PKR的非沃森克里克rna的详细机制和结构。上述机械实验的结果将促进动力学框架的发展，在此框架内分配和理解结构rna的各种作用。这将通过停流实验、平衡荧光研究和rna -蛋白结构-功能分析来实现。RNA结构也将通过结构作图、交联、足迹和几种新的体外选择方法进行检查。对调节PKR功能至关重要的特殊RNA将被选择和富集，目的是确定一套规则，以便预测病毒或细胞RNA是PKR的正调节因子还是负调节因子。

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.