Counting of six pRNAs of phi29 DNA-packaging motor with customized single-molecule dual-view system.
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.
Shu
Dan
D
Department of Comparative Pathobiology and Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907, USA.
Zhang
Hui
H
Jin
Jiashun
J
Guo
Peixuan
P
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*
D004279:DNA, Viral; D046912:Multiprotein Complexes; D012367:RNA, Viral; D005293:Ferritins