A three-helix junction is the interface between two functional domains of prohead RNA in φ29 DNA packaging

Wei Zhao, Mitul Saha, Ailong Ke, Marc Morais, Paul J. Jardine, Shelley Grimes

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

The double-stranded-DNA bacteriophages employ powerful molecular motors to translocate genomic DNA into preformed capsids during the packaging step in phage assembly. Bacillus subtilis bacteriophage φ29 has an oligomeric prohead RNA (pRNA)that is an essential component of its packaging motor. The crystal structure of the pRNA-prohead binding domain suggested that a three-helix junction constitutes both a flexible region and part of a rigid RNA superhelix. Here we definethe functional role of the three-helix junction in motor assembly and DNA packaging. Deletion mutagenesis showed thata U-rich region comprising two sides of the junction plays a role in the stable assembly of pRNA to the prohead. The retention of at least two bulged residues in this region was essential for pRNA binding and thereby subsequent DNA packaging.Additional deletions resulted in the loss of the ability of pRNA to multimerize in solution, consistent with the hypothesis that this region provides the flexibility required for pRNA oligomerization and prohead binding. The third side ofthe junction is part of a large RNA superhelix that spans the motor. The insertion of bases into this feature resulted ina loss of DNA packaging and an impairment of initiation complex assembly. Additionally, cryo-electron microscopy (cryoEM) analysis of third-side insertion mutants showed an increased flexibility of the helix that binds the ATPase, suggesting that the rigidity of the RNA superhelix is necessary for efficient motor assembly and function. These results highlight the critical role of the three-way junction in bridging the prohead binding and ATPase assembly functions of pRNA.

Original languageEnglish (US)
Pages (from-to)11625-11632
Number of pages8
JournalJournal of Virology
Volume86
Issue number21
DOIs
StatePublished - Nov 2012

Fingerprint

DNA Packaging
RNA
Product Packaging
Bacteriophages
Adenosine Triphosphatases
bacteriophages
packaging
adenosinetriphosphatase
Bacillus Phages
Cryoelectron Microscopy
DNA packaging
Capsid
DNA
capsid
Bacillus subtilis
Mutagenesis
crystal structure
mutagenesis

ASJC Scopus subject areas

  • Immunology
  • Virology

Cite this

A three-helix junction is the interface between two functional domains of prohead RNA in φ29 DNA packaging. / Zhao, Wei; Saha, Mitul; Ke, Ailong; Morais, Marc; Jardine, Paul J.; Grimes, Shelley.

In: Journal of Virology, Vol. 86, No. 21, 11.2012, p. 11625-11632.

Research output: Contribution to journalArticle

Zhao, Wei ; Saha, Mitul ; Ke, Ailong ; Morais, Marc ; Jardine, Paul J. ; Grimes, Shelley. / A three-helix junction is the interface between two functional domains of prohead RNA in φ29 DNA packaging. In: Journal of Virology. 2012 ; Vol. 86, No. 21. pp. 11625-11632.
@article{b2b5f0f06f014a879c79296ec455a6bd,
title = "A three-helix junction is the interface between two functional domains of prohead RNA in φ29 DNA packaging",
abstract = "The double-stranded-DNA bacteriophages employ powerful molecular motors to translocate genomic DNA into preformed capsids during the packaging step in phage assembly. Bacillus subtilis bacteriophage φ29 has an oligomeric prohead RNA (pRNA)that is an essential component of its packaging motor. The crystal structure of the pRNA-prohead binding domain suggested that a three-helix junction constitutes both a flexible region and part of a rigid RNA superhelix. Here we definethe functional role of the three-helix junction in motor assembly and DNA packaging. Deletion mutagenesis showed thata U-rich region comprising two sides of the junction plays a role in the stable assembly of pRNA to the prohead. The retention of at least two bulged residues in this region was essential for pRNA binding and thereby subsequent DNA packaging.Additional deletions resulted in the loss of the ability of pRNA to multimerize in solution, consistent with the hypothesis that this region provides the flexibility required for pRNA oligomerization and prohead binding. The third side ofthe junction is part of a large RNA superhelix that spans the motor. The insertion of bases into this feature resulted ina loss of DNA packaging and an impairment of initiation complex assembly. Additionally, cryo-electron microscopy (cryoEM) analysis of third-side insertion mutants showed an increased flexibility of the helix that binds the ATPase, suggesting that the rigidity of the RNA superhelix is necessary for efficient motor assembly and function. These results highlight the critical role of the three-way junction in bridging the prohead binding and ATPase assembly functions of pRNA.",
author = "Wei Zhao and Mitul Saha and Ailong Ke and Marc Morais and Jardine, {Paul J.} and Shelley Grimes",
year = "2012",
month = "11",
doi = "10.1128/JVI.01370-12",
language = "English (US)",
volume = "86",
pages = "11625--11632",
journal = "Journal of Virology",
issn = "0022-538X",
publisher = "American Society for Microbiology",
number = "21",

}

TY - JOUR

T1 - A three-helix junction is the interface between two functional domains of prohead RNA in φ29 DNA packaging

AU - Zhao, Wei

AU - Saha, Mitul

AU - Ke, Ailong

AU - Morais, Marc

AU - Jardine, Paul J.

AU - Grimes, Shelley

PY - 2012/11

Y1 - 2012/11

N2 - The double-stranded-DNA bacteriophages employ powerful molecular motors to translocate genomic DNA into preformed capsids during the packaging step in phage assembly. Bacillus subtilis bacteriophage φ29 has an oligomeric prohead RNA (pRNA)that is an essential component of its packaging motor. The crystal structure of the pRNA-prohead binding domain suggested that a three-helix junction constitutes both a flexible region and part of a rigid RNA superhelix. Here we definethe functional role of the three-helix junction in motor assembly and DNA packaging. Deletion mutagenesis showed thata U-rich region comprising two sides of the junction plays a role in the stable assembly of pRNA to the prohead. The retention of at least two bulged residues in this region was essential for pRNA binding and thereby subsequent DNA packaging.Additional deletions resulted in the loss of the ability of pRNA to multimerize in solution, consistent with the hypothesis that this region provides the flexibility required for pRNA oligomerization and prohead binding. The third side ofthe junction is part of a large RNA superhelix that spans the motor. The insertion of bases into this feature resulted ina loss of DNA packaging and an impairment of initiation complex assembly. Additionally, cryo-electron microscopy (cryoEM) analysis of third-side insertion mutants showed an increased flexibility of the helix that binds the ATPase, suggesting that the rigidity of the RNA superhelix is necessary for efficient motor assembly and function. These results highlight the critical role of the three-way junction in bridging the prohead binding and ATPase assembly functions of pRNA.

AB - The double-stranded-DNA bacteriophages employ powerful molecular motors to translocate genomic DNA into preformed capsids during the packaging step in phage assembly. Bacillus subtilis bacteriophage φ29 has an oligomeric prohead RNA (pRNA)that is an essential component of its packaging motor. The crystal structure of the pRNA-prohead binding domain suggested that a three-helix junction constitutes both a flexible region and part of a rigid RNA superhelix. Here we definethe functional role of the three-helix junction in motor assembly and DNA packaging. Deletion mutagenesis showed thata U-rich region comprising two sides of the junction plays a role in the stable assembly of pRNA to the prohead. The retention of at least two bulged residues in this region was essential for pRNA binding and thereby subsequent DNA packaging.Additional deletions resulted in the loss of the ability of pRNA to multimerize in solution, consistent with the hypothesis that this region provides the flexibility required for pRNA oligomerization and prohead binding. The third side ofthe junction is part of a large RNA superhelix that spans the motor. The insertion of bases into this feature resulted ina loss of DNA packaging and an impairment of initiation complex assembly. Additionally, cryo-electron microscopy (cryoEM) analysis of third-side insertion mutants showed an increased flexibility of the helix that binds the ATPase, suggesting that the rigidity of the RNA superhelix is necessary for efficient motor assembly and function. These results highlight the critical role of the three-way junction in bridging the prohead binding and ATPase assembly functions of pRNA.

UR - http://www.scopus.com/inward/record.url?scp=84869048081&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84869048081&partnerID=8YFLogxK

U2 - 10.1128/JVI.01370-12

DO - 10.1128/JVI.01370-12

M3 - Article

C2 - 22896620

AN - SCOPUS:84869048081

VL - 86

SP - 11625

EP - 11632

JO - Journal of Virology

JF - Journal of Virology

SN - 0022-538X

IS - 21

ER -