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.
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