Global structure of a three-way junction in a phi29 packaging RNA dimer determined using site-directed spin labeling.
The condensation of bacteriophage phi29 genomic DNA into its preformed procapsid requires the DNA packaging motor, which is the strongest known biological motor. The packaging motor is an intricate ring-shaped protein/RNA complex, and its function requires an RNA component called packaging RNA (pRNA). Current structural information on pRNA is limited, which hinders studies of motor function. Here, we used site-directed spin labeling to map the conformation of a pRNA three-way junction that bridges binding sites for the motor ATPase and the procapsid. The studies were carried out on a pRNA dimer, which is the simplest ring-shaped pRNA complex and serves as a functional intermediate during motor assembly. Using a nucleotide-independent labeling scheme, stable nitroxide radicals were attached to eight specific pRNA sites without perturbing RNA folding and dimer formation, and a total of 17 internitroxide distances spanning the three-way junction were measured using Double Electron-Electron Resonance spectroscopy. The measured distances, together with steric chemical constraints, were used to select 3662 viable three-way junction models from a pool of 65 billion. The results reveal a similar conformation among the viable models, with two of the helices (H(T) and H(L)) adopting an acute bend. This is in contrast to a recently reported pRNA tetramer crystal structure, in which H(T) and H(L) stack onto each other linearly. The studies establish a new method for mapping global structures of complex RNA molecules, and provide information on pRNA conformation that aids investigations of phi29 packaging motor and developments of pRNA-based nanomedicine and nanomaterial.
Department of Chemistry, University of Southern California, Los Angeles, California 90089, USA.
Xiaojun
X
Zhang
Chang-Shung
CS
Tung
Glenna Z
GZ
Sowa
Ma'mon M
MM
Hatmal
Ian S
IS
Haworth
Peter Z
PZ
Qin
D001435:Bacteriophages / Q000737:chemistry; D019281:Dimerization; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000737:chemistry*; D013113:Spin Labels
D012367:RNA, Viral; D013113:Spin Labels