Binding of pRNA to the N-terminal 14 amino acids of connector protein of bacteriophage phi29.
During assembly, bacterial virus phi29 utilizes a motor to insert genomic DNA into a preformed protein shell called the procapsid. The motor contains one twelve-subunit connector with a 3.6 nm central channel for DNA transportation, six viral-encoded RNA (packaging RNA or pRNA) and a protein, gp16, with unknown stoichiometry. Recent DNA-packaging models proposed that the 5-fold procapsid vertexes and 12-fold connector (or the hexameric pRNA ring) represented a symmetry mismatch enabling production of a force to drive a rotation motor to translocate and compress DNA. There was a discrepancy regarding the location of the foothold for the pRNA. One model [C. Chen and P. Guo (1997) J. Virol., 71, 3864-3871] suggested that the foothold for pRNA was the connector and that the pRNA-connector complex was part of the rotor. However, one other model suggested that the foothold for pRNA was the 5-fold vertex of the capsid protein and that pRNA was the stator. To elucidate the mechanism of phi29 DNA packaging, it is critical to confirm whether pRNA binds to the 5-fold vertex of the capsid protein or to the 12-fold symmetrical connector. Here, we used both purified connector and purified procapsid for binding studies with in vitro transcribed pRNA. Specific binding of pRNA to the connector in the procapsid was found by photoaffinity crosslinking. Removal of the N-terminal 14 amino acids of the gp10 protein by proteolytic cleavage resulted in undetectable binding of pRNA to either the connector or the procapsid, as investigated by agarose gel electrophoresis, SDS-PAGE, sucrose gradient sedimentation and N-terminal peptide sequencing. It is therefore concluded that pRNA bound to the 12-fold symmetrical connector to form a pRNA-connector complex and that the foothold for pRNA is the connector but not the capsid protein.
Xiao
Feng
F
Department of Pathobiology, Wildon School of Bioengineering and Purdue Cancer Research Center, Purdue University, West Lafayette, IN 47907, USA.
Moll
Wulf-Dieter
WD
Guo
Songchuan
S
Guo
Peixuan
P
D000595:Amino Acid Sequence; D017103:Bacillus Phages / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D001483:Base Sequence; D001665:Binding Sites; D036022:Capsid Proteins / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D024202:Electrophoretic Mobility Shift Assay; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D019065:Virus Assembly
D036022:Capsid Proteins; D012367:RNA, Viral; C097975:portal protein, bacteriophage phi29