Crystal structure of BstDEAD, a novel DEAD-box protein from Bacillus stearothermophilus

BstDEAD is a representative member of the DEAD-protein family from the thermophilic bacterium Bacillus stearothermophilus. The BstDEAD core contains all nine helicase signature sequences that are conserved among DEAD-box proteins, plus a unique C-terminal extension that may denote a specific biochemical role. Sequence homology with other characterized members of the DEAD-protein family suggests that BstDEAD is a putative RNA helicase.; DEAD-box proteins belong to a large and diverse family whose members are involved in most biochemical processes involving RNA. DEAD-box proteins possess RNA-dependent ATPase activity and most are believed to unwind regions of duplex RNA. In addition, published structures of both RNA and DNA helicases imply that the core helicase domains are structurally conserved.; A high-resolution structure of the N-terminal domain of BstDEAD (BstDEAD-NT) and a low-resolution structure of full-length BstDEAD were solved using X-ray crystallography. The core domains appear structurally similar to those of closely related DEAD-box proteins and to the corresponding domains in more distantly related DNA helicases. The BstDEAD-NT structure defines a cleft formed by the conserved residues responsible for binding and hydrolysis of ATP. The overall domain architecture is detailed in the full-length structure along with a portion of the unique C-terminal extension.; While the precise biological role of BstDEAD remains unknown, biochemical experiments demonstrated that BstDEAD has characteristically low nucleic acid-independent ATPase activity that is stimulated in the presence of RNA. The extent of stimulation depends on the particular RNA examined. Both ultracentrifugation experiments and the crystal structure of full-length BstDEAD suggest that a BstDEAD monomer is the relevant state for biological activity. In addition, while BstDEAD binds RNA with high affinity, likely mediated by basic residues within the C-terminal extension, duplex RNA unwinding was not observed.; This dissertation describes the crystal structure of BstDEAD-NT and the structural and biochemical characterization of full-length BstDEAD. Also included is a description of previous attempts to crystallize other protein-RNA complexes. This dissertation includes my co-authored materials.