Talk abstracts

Talk on Friday 09:50-10:10pm submitted by Tae Jin Lee

Interaction with pRNA and DNA-dependent ATP hydrolysis of gp16 of bacterial virus phi29 DNA packaging motor

Tae Jin Lee (Department of Biomedical Engineering, University of Cincinnati), Hui Zhang (Department of Biomedical Engineering, University of Cincinnati), Peixuan Guo (Department of Biomedical Engineering, University of Cincinnati)

Abstract:
All linear dsDNA viruses, including bacteriophage phi29, translocate their genome into a pre-assembled procapsid to near-crystalline density. The packaging enzyme in the viral DNA-packaging motor generates the driving force to overcome this entropically unfavorable packaging reaction. We investigated the specific interaction of the putative packaging enzyme of the phi29 packaging motor with packaging RNA (pRNA).
Our biochemical studies shows that the 5'/3' paired helical region on pRNA in the pRNA/procapsid complex serves as a docking site for gp16 binding. Gp16 bound to the pRNA-containing procapsid much more strongly than to the pRNA-free procapsid. The pRNA bulge C18C19A20 that is essential for DNA packaging was found to be dispensable for gp16 binding. These results imply that pRNA, with its central domain binds to the procapsid, extends its 5'/3' DNA-packaging domain for gp16 interaction. The interaction of gp16 with nucleic acids was also investigated using recently developed single molecule system. The technique employs TIRF enables to observe the binding between molecules. The study showed that gp16 binds nucleic acids (DNA or RNA) depending on their structures and chemistry. In addition, In vitro binding assay showed that gp16 binds to dsDNA in a non-sequence specific manner.
Gp16 contains a conserved nucleotide triphosphate binding motif. The steady-state analysis showed that the nucleic acids binding stimulates the ATP hydrolysis of gp16, and the extent is also dependent on the structure and chemistry of DNA or RNA. From the data, the kinetic parameters including Km and the rate constant for ATP hydrolysis were determined. Our results strongly suggest that gp16 interacts with nucleic acids including pRNA as a part of ATP hydrolyzing complex in the packaging motor, and its ATPase activity can be stimulated via those interactions.

Keywords: Bacteriophage phi29 packaing motor, Packaging RNA, Single molecule imaging