This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The objective of this project is to determine the multimeric state of the prohead RNA (pRNA) of bacteriophage 29 DNA packaging motor.pRNA of the Bacillus subtilis bacteriophage 29, a 174-base transcript of the phage genome, is an essential component of the 29 DNA packaging motor. Multiple copies of pRNA bind to the head-tail connector of the precursor protein shell (prohead). Subsequent binding of the ATPase gp16 to pRNA constitutes the motor that translocates the 19 kilobase pair DNA-gene product 3 complex (DNA-gp3). Genetic experiments have suggested that identical pRNA molecules are linked through pseudoknots resulting in a hexameric structure [1, 2]. However, cryoEM-3D reconstruction has shown that pRNA has 5-fold symmetry when it attached to the prohead [3-5]. We have identified intermolecular hydrogen bonds within the pseudoknot of pRNA by using NMR spectroscopy, and are currently determining the three dimensional structure of the pseudoknot of pRNA, which represents a new structural motif in the RNA world. We will obtain relative orientation of individual pRNA molecules in the ring by measuring residual dipolar couplings using NMR. We need to perform SAXS experiments to verify how many pRNA molecules are in the ring structure. By combining the pseudoknot structure, the alignment of individual molecules and the number of molecules in the ring, we will build the three dimensional structure of the pRNA ring.
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