? DExH/D proteins are involved in virtually all aspects of RNA metabolism in the cell and in many viruses. Numerous proteins from this family have been shown to play direct roles in disease states such as in tumorigenesis and in the replication of the hepatitis C and the smallpox virus. DExH/D proteins unwind RNA structures (RNA helicase) and/or re-arrange RNA-protein complexes (RNPase) in an ATP-dependent fashion. Despite the biological importance of DExH/D proteins, their mechanism of action is not understood, mainly due to the highly complex nature of the reactions. We propose to gain essential insight into these questions by employing single molecule fluorescence in conjunction with biochemical approaches to investigate the prototypical DExH/D protein NPH-II from vaccinia virus. ? ? First, we will develop a mechanistic framework for RNA helicase activity at the single molecule level to address the fundamental question the how DExH/D proteins use ATP to effect conformational changes in RNA. Using single molecule fluorescence energy transfer (FRET) we will determine how ATP binding and hydrolysis is coupled to conformational changes in the RNA, to the oligomeric state of NPH-II, and to the translocation of NPH-II during duplex unwinding. ? ? Second, we will probe translocation of NPH-II along single stranded RNA. It has been hypothesized that an ability to translocate along single stranded RNA could give rise to the multiple activities of DExH/D proteins such as duplex unwinding and remodeling of RNA protein complexes. Yet, translocation along single stranded RNA has never been tested. We will directly test whether NPH-II translocates along single stranded RNA using single molecule FRET. ? ? Third, we will investigate the physical basis of RNPase activity. Employing a biochemical approach, we will test whether DExH/D proteins displace other proteins from RNA through direct physical contact, or through induction of torsional strain in the RNA to flip other proteins off the RNA. In addition we will investigate how NPH-II couples ATP binding and hydrolysis to the remodeling of RNA-protein complexes. ? ?
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