Poster abstracts
Poster number 7 submitted by Margaret Bohmer
Constructing an active chimeric pRNA ring with a stoichiometry of six and identifying 12 domains of the pRNA ring binding to the 12-subunit channel of phi29 DNA packaging motor
Margaret Bohmer (College of Pharmacy, Division of Pharmaceutics and Pharmacology; Center for RNA Biology; The Ohio State University, Columbus, Ohio, USA.), Daniel W Binzel (College of Pharmacy, Division of Pharmaceutics and Pharmacology; Center for RNA Nanobiotechnology and Nanomedicine; James Comprehensive Cancer Center; The Ohio State University, Columbus, Ohio, USA.), Wen Zhang (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN SA), Peixuan Guo (College of Pharmacy, Division of Pharmaceutics and Pharmacology; Center for RNA Nanobiotechnology and Nanomedicine; James Comprehensive Cancer Center; The Ohio State University, Columbus, Ohio, USA.)
Abstract:
During the last stage of replication of double-stranded RNA or DNA viruses, their genome is packaged into a preassembled protein capsid. The bacterial virus phi29 dsDNA packaging motor uses a noncoding packaging RNA (pRNA) molecule to gear its genomic DNA translocation. In this study, we constructed chimeric pRNAs by fusing the pRNA of bacterial virus M2 and that of phi29. The chimeric pRNAs can form dimers or trimers. The dimeric or trimeric pRNAs were active in the packaging of the phi29 dsDNA genome into the purified procapsid, which was subsequently converted into the infectious viruses, as proven by counting plaque-forming-units (PFU). These data show that the stoichiometry of the chimeric pRNAs on the motor is 6 subunits, a multiple of 2 and 3. Furthermore, AFM studies on pRNA fused to an RNA-triangle revealed hexamer formation. But how do the six identical RNA anchor on the 12-subunit connector with the double stoichiometry? Structural analysis in combination with enzymatic and chemical probing data revealed that each native pRNA contributes 2 domains to bind to the 12-subunit DNA-packaging channel at 3 positively charged residues RKR, proving the formation of the hexameric ring. Resolving the hexamer versus pentamer debate clarifies the mechanism of dsDNA translocation in living organisms.
References:
Bohmer M, Binzel DW, Zhang W, Guo P. Constructing an active chimeric pRNA ring with a stoichiometry of six and identifying 12 domains of the pRNA ring binding to the 12-subunit channel of phi29 DNA packaging motor. RNA. 2025 Apr 1:rna.080383.125. doi: 10.1261/rna.080383.125. Epub ahead of print. PMID: 40169225.
Keywords: Revolving motor, Hexameric ATPase, Asymmetrical biomotor