DNA Packaging Motor Assembly Intermediate of Bacteriophage φ{symbol}29

Jaya S. Koti, Marc Morais, Raj Rajagopal, Barbara A L Owen, Cynthia T. McMurray, Dwight L. Anderson

Research output: Contribution to journalArticle

24 Citations (Scopus)

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 φ{symbol}29 packaging ATPase gene product 16 (gp16) was overexpressed in soluble form in Bacillus subtilis (pSAC), purified to near homogeneity, and assembled to the φ{symbol}29 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.

Original languageEnglish (US)
Pages (from-to)1114-1132
Number of pages19
JournalJournal of Molecular Biology
Volume381
Issue number5
DOIs
StatePublished - Sep 19 2008
Externally publishedYes

Fingerprint

DNA Packaging
Bacteriophages
Product Packaging
Genes
RNA
DNA
Adenosine Triphosphatases
Cryoelectron Microscopy
Efferent Pathways
Capsid
Ultracentrifugation
Ribonucleases
Bacillus subtilis
Gel Chromatography
Electron Microscopy
Head

Keywords

  • ATPase gp16
  • bacteriophage φ{symbol}29
  • DNA packaging
  • phage assembly
  • RNA binding

ASJC Scopus subject areas

  • Virology

Cite this

Koti, J. S., Morais, M., Rajagopal, R., Owen, B. A. L., McMurray, C. T., & Anderson, D. L. (2008). DNA Packaging Motor Assembly Intermediate of Bacteriophage φ{symbol}29. Journal of Molecular Biology, 381(5), 1114-1132. https://doi.org/10.1016/j.jmb.2008.04.034

DNA Packaging Motor Assembly Intermediate of Bacteriophage φ{symbol}29. / Koti, Jaya S.; Morais, Marc; Rajagopal, Raj; Owen, Barbara A L; McMurray, Cynthia T.; Anderson, Dwight L.

In: Journal of Molecular Biology, Vol. 381, No. 5, 19.09.2008, p. 1114-1132.

Research output: Contribution to journalArticle

Koti, JS, Morais, M, Rajagopal, R, Owen, BAL, McMurray, CT & Anderson, DL 2008, 'DNA Packaging Motor Assembly Intermediate of Bacteriophage φ{symbol}29', Journal of Molecular Biology, vol. 381, no. 5, pp. 1114-1132. https://doi.org/10.1016/j.jmb.2008.04.034
Koti, Jaya S. ; Morais, Marc ; Rajagopal, Raj ; Owen, Barbara A L ; McMurray, Cynthia T. ; Anderson, Dwight L. / DNA Packaging Motor Assembly Intermediate of Bacteriophage φ{symbol}29. In: Journal of Molecular Biology. 2008 ; Vol. 381, No. 5. pp. 1114-1132.
@article{db5e596b7d1e49689d29337c28687c04,
title = "DNA Packaging Motor Assembly Intermediate of Bacteriophage φ{symbol}29",
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 φ{symbol}29 packaging ATPase gene product 16 (gp16) was overexpressed in soluble form in Bacillus subtilis (pSAC), purified to near homogeneity, and assembled to the φ{symbol}29 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.",
keywords = "ATPase gp16, bacteriophage φ{symbol}29, DNA packaging, phage assembly, RNA binding",
author = "Koti, {Jaya S.} and Marc Morais and Raj Rajagopal and Owen, {Barbara A L} and McMurray, {Cynthia T.} and Anderson, {Dwight L.}",
year = "2008",
month = "9",
day = "19",
doi = "10.1016/j.jmb.2008.04.034",
language = "English (US)",
volume = "381",
pages = "1114--1132",
journal = "Journal of Molecular Biology",
issn = "0022-2836",
publisher = "Academic Press Inc.",
number = "5",

}

TY - JOUR

T1 - DNA Packaging Motor Assembly Intermediate of Bacteriophage φ{symbol}29

AU - Koti, Jaya S.

AU - Morais, Marc

AU - Rajagopal, Raj

AU - Owen, Barbara A L

AU - McMurray, Cynthia T.

AU - Anderson, Dwight L.

PY - 2008/9/19

Y1 - 2008/9/19

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 φ{symbol}29 packaging ATPase gene product 16 (gp16) was overexpressed in soluble form in Bacillus subtilis (pSAC), purified to near homogeneity, and assembled to the φ{symbol}29 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.

AB - 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 φ{symbol}29 packaging ATPase gene product 16 (gp16) was overexpressed in soluble form in Bacillus subtilis (pSAC), purified to near homogeneity, and assembled to the φ{symbol}29 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.

KW - ATPase gp16

KW - bacteriophage φ{symbol}29

KW - DNA packaging

KW - phage assembly

KW - RNA binding

UR - http://www.scopus.com/inward/record.url?scp=48749126443&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=48749126443&partnerID=8YFLogxK

U2 - 10.1016/j.jmb.2008.04.034

DO - 10.1016/j.jmb.2008.04.034

M3 - Article

VL - 381

SP - 1114

EP - 1132

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 5

ER -