Strand and nucleotide-dependent ATPase activity of gp16 of bacterial virus phi29 DNA packaging motor.
Similar to the assembly of other dsDNA viruses, bacterial virus phi29 uses a motor to translocate its DNA into a procapsid, with the aid of protein gp16 that binds to pRNA 5'/3' helical region. To investigate the mechanism of the motor action, the kinetics of the ATPase activity of gp16 was evaluated as a function of DNA structure (ss- or ds-stranded) or chemistry (purine or pyrimidine). The k(cat) and K(m) in the absence of DNA was 0.016 s(-1) and 351.0 microM, respectively, suggesting that gp16 itself is a slow-ATPase with a low affinity for substrate. The affinity of gp16 for ATP was greatly boosted by the presence of DNA or pRNA, but the ATPase rate was strongly affected by DNA structure and chemistry. The order of ATPase stimulation is poly d(pyrimidine)>dsDNA>poly d(purine), which agreed with the order of the DNA binding to gp16, as revealed by single molecule fluorescence microscopy. Interestingly, the stimulation degree by phi29 pRNA was similar to that of poly d(pyrimidine). The results suggest that pRNA accelerates gp16 ATPase activity more significantly than genomic dsDNA, albeit both pRNA and genomic DNA are involved in the contact with gp16 during DNA packaging.
Lee
Tae Jin
TJ
Department of Biomedical Engineering, College of Engineering and College of Medicine, University of Cincinnati, Cincinnati, OH 45267, USA.
Zhang
Hui
H
Liang
Dan
D
Guo
Peixuan
P
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D017103:Bacillus Phages / Q000235:genetics / Q000502:physiology*; D001665:Binding Sites; D002213:Capsid / Q000378:metabolism; D042003:DNA Packaging / Q000502:physiology*; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D012367:RNA, Viral / Q000378:metabolism*; D019065:Virus Assembly / Q000235:genetics / Q000502:physiology
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D012367:RNA, Viral; D000251:Adenosine Triphosphatases; C439525:protein p16, bacteriophage phi29