Cryo-electron tomography of bacteriophage phi6 procapsids shows random occupancy of the binding sites for RNA polymerase and packaging NTPase.
Assembly of dsRNA bacteriophage phi6 involves packaging of the three mRNA strands of the segmented genome into the procapsid, an icosahedrally symmetric particle with recessed vertices. The hexameric packaging NTPase (P4) overlies these vertices, and the monomeric RNA-dependent RNA polymerase (RdRP, P2) binds at sites inside the shell. P2 and P4 are present in substoichiometric amounts, raising the questions of whether they are recruited to the nascent procapsid in defined amounts and at specific locations, and whether they may co-localize to form RNA-processing assembly lines at one or more "special" vertices. We have used cryo-electron tomography to map both molecules on individual procapsids. The results show variable complements that accord with binomial distributions with means of 8 (P2) and 5 (P4), suggesting that they are randomly incorporated in copy numbers that simply reflect availability, i.e. their rates of synthesis. Analysis of the occupancy of potential binding sites (20 for P2; 12 for P4) shows no tendency to cluster nor for P2 and P4 to co-localize, suggesting that the binding sites for both proteins are occupied in random fashion. These observations indicate that although P2 and P4 act sequentially on the same substrates there is no direct physical coupling between their activities.
National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, 50 South Dr, Bethesda, MD 20892, USA.
Daniel
D
Nemecek
J Bernard
JB
Heymann
Jian
J
Qiao
Leonard
L
Mindich
Alasdair C
AC
Steven
D017126:Bacteriophage phi 6 / Q000378:metabolism* / Q000648:ultrastructure*; D001665:Binding Sites; D002213:Capsid / Q000378:metabolism* / Q000648:ultrastructure*; D020285:Cryoelectron Microscopy; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D055032:Electron Microscope Tomography; D043583:Nucleoside-Triphosphatase / Q000378:metabolism*
D012321:DNA-Directed RNA Polymerases; D043583:Nucleoside-Triphosphatase