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DNA packaging motor assembly intermediate of bacteriophage phi29.
J Mol Biol 2008; 381(5):1114-32JM

Abstract

Unraveling the structure and assembly of the DNA packaging ATPases of the tailed double-stranded DNA bacteriophages is integral to understanding the mechanism of DNA translocation. Here, the bacteriophage phi29 packaging ATPase gene product 16 (gp16) was overexpressed in soluble form in Bacillus subtilis (pSAC), purified to near homogeneity, and assembled to the phi29 precursor capsid (prohead) to produce a packaging motor intermediate that was fully active in in vitro DNA packaging. The formation of higher oligomers of the gp16 from monomers was concentration dependent and was characterized by analytical ultracentrifugation, gel filtration, and electron microscopy. The binding of multiple copies of gp16 to the prohead was dependent on the presence of an oligomer of 174- or 120-base prohead RNA (pRNA) fixed to the head-tail connector at the unique portal vertex of the prohead. The use of mutant pRNAs demonstrated that gp16 bound specifically to the A-helix of pRNA, and ribonuclease footprinting of gp16 on pRNA showed that gp16 protected the CC residues of the CCA bulge (residues 18-20) of the A-helix. The binding of gp16 to the prohead/pRNA to constitute the complete and active packaging motor was confirmed by cryo-electron microscopy three-dimensional reconstruction of the prohead/pRNA/gp16 complex. The complex was capable of supercoiling DNA-gp3 as observed previously for gp16 alone; therefore, the binding of gp16 to the prohead, rather than first to DNA-gp3, represents an alternative packaging motor assembly pathway.

Authors+Show Affiliations

Department of Diagnostic/Biological Sciences, University of Minnesota, Minneapolis, MN 55455, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

18674782

Citation

Koti, Jaya S., et al. "DNA Packaging Motor Assembly Intermediate of Bacteriophage Phi29." Journal of Molecular Biology, vol. 381, no. 5, 2008, pp. 1114-32.
Koti JS, Morais MC, Rajagopal R, et al. DNA packaging motor assembly intermediate of bacteriophage phi29. J Mol Biol. 2008;381(5):1114-32.
Koti, J. S., Morais, M. C., Rajagopal, R., Owen, B. A., McMurray, C. T., & Anderson, D. L. (2008). DNA packaging motor assembly intermediate of bacteriophage phi29. Journal of Molecular Biology, 381(5), pp. 1114-32. doi:10.1016/j.jmb.2008.04.034.
Koti JS, et al. DNA Packaging Motor Assembly Intermediate of Bacteriophage Phi29. J Mol Biol. 2008 Sep 19;381(5):1114-32. PubMed PMID: 18674782.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - DNA packaging motor assembly intermediate of bacteriophage phi29. AU - Koti,Jaya S, AU - Morais,Marc C, AU - Rajagopal,Raj, AU - Owen,Barbara A L, AU - McMurray,Cynthia T, AU - Anderson,Dwight L, Y1 - 2008/04/20/ PY - 2008/03/01/received PY - 2008/04/10/revised PY - 2008/04/11/accepted PY - 2008/8/5/pubmed PY - 2008/9/16/medline PY - 2008/8/5/entrez SP - 1114 EP - 32 JF - Journal of molecular biology JO - J. Mol. Biol. VL - 381 IS - 5 N2 - Unraveling the structure and assembly of the DNA packaging ATPases of the tailed double-stranded DNA bacteriophages is integral to understanding the mechanism of DNA translocation. Here, the bacteriophage phi29 packaging ATPase gene product 16 (gp16) was overexpressed in soluble form in Bacillus subtilis (pSAC), purified to near homogeneity, and assembled to the phi29 precursor capsid (prohead) to produce a packaging motor intermediate that was fully active in in vitro DNA packaging. The formation of higher oligomers of the gp16 from monomers was concentration dependent and was characterized by analytical ultracentrifugation, gel filtration, and electron microscopy. The binding of multiple copies of gp16 to the prohead was dependent on the presence of an oligomer of 174- or 120-base prohead RNA (pRNA) fixed to the head-tail connector at the unique portal vertex of the prohead. The use of mutant pRNAs demonstrated that gp16 bound specifically to the A-helix of pRNA, and ribonuclease footprinting of gp16 on pRNA showed that gp16 protected the CC residues of the CCA bulge (residues 18-20) of the A-helix. The binding of gp16 to the prohead/pRNA to constitute the complete and active packaging motor was confirmed by cryo-electron microscopy three-dimensional reconstruction of the prohead/pRNA/gp16 complex. The complex was capable of supercoiling DNA-gp3 as observed previously for gp16 alone; therefore, the binding of gp16 to the prohead, rather than first to DNA-gp3, represents an alternative packaging motor assembly pathway. SN - 1089-8638 UR - https://www.unboundmedicine.com/medline/citation/18674782/DNA_packaging_motor_assembly_intermediate_of_bacteriophage_phi29_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0022-2836(08)00466-X DB - PRIME DP - Unbound Medicine ER -