title: Counting of six pRNAs of phi29 DNA-packaging motor with customized single-molecule dual-view system.
abstract: Direct imaging or counting of RNA molecules has been difficult owing to its relatively low electron density for EM and insufficient resolution in AFM. Bacteriophage phi29 DNA-packaging motor is geared by a packaging RNA (pRNA) ring. Currently, whether the ring is a pentagon or hexagon is under fervent debate. We report here the assembly of a highly sensitive imaging system for direct counting of the copy number of pRNA within this 20-nm motor. Single fluorophore imaging clearly identified the quantized photobleaching steps from pRNA labeled with a single fluorophore and concluded its stoichiometry within the motor. Almost all of the motors contained six copies of pRNA before and during DNA translocation, identified by dual-color detection of the stalled intermediates of motors containing Cy3-pRNA and Cy5-DNA. The stalled motors were restarted to observe the motion of DNA packaging in real time. Heat-denaturation analysis confirmed that the stoichiometry of pRNA is the common multiple of 2 and 3. EM imaging of procapsid/pRNA complexes clearly revealed six ferritin particles that were conjugated to each pRNA ring.
authors.affiliation: Department of Comparative Pathobiology and Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Dan
authors.initials: D
authors.lastname: Shu
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.forename: Jiashun
authors.initials: J
authors.lastname: Jin
authors.forename: Peixuan
authors.initials: P
authors.lastname: Guo
mesh_terms: D017103:Bacillus Phages / Q000648:ultrastructure*; D002213:Capsid / Q000737:chemistry; D042003:DNA Packaging; D004279:DNA, Viral; D019281:Dimerization; D005293:Ferritins / Q000737:chemistry; D006358:Hot Temperature; D008854:Microscopy, Electron / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods; D008954:Models, Biological; D046912:Multiprotein Complexes / Q000737:chemistry; D012367:RNA, Viral / Q000502:physiology*
chemical_list: D004279:DNA, Viral; D046912:Multiprotein Complexes; D012367:RNA, Viral; D005293:Ferritins