Alanine Scanning and Fe-BABE Probing of the Bacteriophage ø29 Prohead RNA-Connector Interaction

Rockney Atz; Shuhua Ma; Jiali Gao; Dwight L. Anderson; Shelley Grimes

doi:10.1016/j.jmb.2007.03.033

Alanine Scanning and Fe-BABE Probing of the Bacteriophage ø29 Prohead RNA-Connector Interaction

Rockney Atz
, Shuhua Ma
, Jiali Gao
, Dwight L. Anderson
, Shelley Grimes

Research output: Contribution to journal › Article › peer-review

25 Scopus citations

Abstract

The DNA packaging motor of the Bacillus subtilis bacteriophage ø29 prohead is comprised in part of an oligomeric ring of 174 base RNA molecules (pRNA) positioned near the N termini of subunits of the dodecameric head-tail connector. Deletion and alanine substitution mutants in the connector protein (gp10) N terminus were assembled into proheads in Escherichia coli and the particles tested for pRNA binding and DNA-gp3 packaging in vitro. The basic amino acid residues RKR at positions 3-5 of the gp10 N terminus were central to pRNA binding during assembly of an active DNA packaging motor. Conjugation of iron(S)-1-(p-bromoacetamidobenzyl) ethylenediaminetetraacetate (Fe-BABE) to residue S170C in the narrow end of the connector, near the N terminus, permitted hydroxyl radical probing of bound [³²P]pRNA and identified two discrete sites proximal to this residue: the C-helix at the junction of the A, C and D helices, and the E helix and the CE loop/D loop of the intermolecular base pairing site.

Original language	English (US)
Pages (from-to)	239-248
Number of pages	10
Journal	Journal of Molecular Biology
Volume	369
Issue number	1
DOIs	https://doi.org/10.1016/j.jmb.2007.03.033
State	Published - May 25 2007

Bibliographical note

Funding Information:
We thank Jaya Koti and Laura Birkeland for preparing 120nt pRNA, Marc Morais for Figure 1 (c), Paul Jardine for helpful discussion, and Charlene Peterson for assistance in the preparation of the manuscript. This research was supported by NIH grants GM-059604, DE-03606 (to S.G.) and GM-46736 (to J.G.).

Keywords

DNA packaging
RNA binding
bacteriophage ø29
directed hydroxyl radical probing
packaging motor assembly

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10.1016/j.jmb.2007.03.033

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title = "Alanine Scanning and Fe-BABE Probing of the Bacteriophage {\o}29 Prohead RNA-Connector Interaction",

abstract = "The DNA packaging motor of the Bacillus subtilis bacteriophage {\o}29 prohead is comprised in part of an oligomeric ring of 174 base RNA molecules (pRNA) positioned near the N termini of subunits of the dodecameric head-tail connector. Deletion and alanine substitution mutants in the connector protein (gp10) N terminus were assembled into proheads in Escherichia coli and the particles tested for pRNA binding and DNA-gp3 packaging in vitro. The basic amino acid residues RKR at positions 3-5 of the gp10 N terminus were central to pRNA binding during assembly of an active DNA packaging motor. Conjugation of iron(S)-1-(p-bromoacetamidobenzyl) ethylenediaminetetraacetate (Fe-BABE) to residue S170C in the narrow end of the connector, near the N terminus, permitted hydroxyl radical probing of bound [32P]pRNA and identified two discrete sites proximal to this residue: the C-helix at the junction of the A, C and D helices, and the E helix and the CE loop/D loop of the intermolecular base pairing site.",

keywords = "DNA packaging, RNA binding, bacteriophage {\o}29, directed hydroxyl radical probing, packaging motor assembly",

author = "Rockney Atz and Shuhua Ma and Jiali Gao and Anderson, \{Dwight L.\} and Shelley Grimes",

note = "Funding Information: We thank Jaya Koti and Laura Birkeland for preparing 120nt pRNA, Marc Morais for Figure 1 (c), Paul Jardine for helpful discussion, and Charlene Peterson for assistance in the preparation of the manuscript. This research was supported by NIH grants GM-059604, DE-03606 (to S.G.) and GM-46736 (to J.G.).",

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AU - Ma, Shuhua

AU - Gao, Jiali

AU - Anderson, Dwight L.

AU - Grimes, Shelley

N1 - Funding Information: We thank Jaya Koti and Laura Birkeland for preparing 120nt pRNA, Marc Morais for Figure 1 (c), Paul Jardine for helpful discussion, and Charlene Peterson for assistance in the preparation of the manuscript. This research was supported by NIH grants GM-059604, DE-03606 (to S.G.) and GM-46736 (to J.G.).

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N2 - The DNA packaging motor of the Bacillus subtilis bacteriophage ø29 prohead is comprised in part of an oligomeric ring of 174 base RNA molecules (pRNA) positioned near the N termini of subunits of the dodecameric head-tail connector. Deletion and alanine substitution mutants in the connector protein (gp10) N terminus were assembled into proheads in Escherichia coli and the particles tested for pRNA binding and DNA-gp3 packaging in vitro. The basic amino acid residues RKR at positions 3-5 of the gp10 N terminus were central to pRNA binding during assembly of an active DNA packaging motor. Conjugation of iron(S)-1-(p-bromoacetamidobenzyl) ethylenediaminetetraacetate (Fe-BABE) to residue S170C in the narrow end of the connector, near the N terminus, permitted hydroxyl radical probing of bound [32P]pRNA and identified two discrete sites proximal to this residue: the C-helix at the junction of the A, C and D helices, and the E helix and the CE loop/D loop of the intermolecular base pairing site.

AB - The DNA packaging motor of the Bacillus subtilis bacteriophage ø29 prohead is comprised in part of an oligomeric ring of 174 base RNA molecules (pRNA) positioned near the N termini of subunits of the dodecameric head-tail connector. Deletion and alanine substitution mutants in the connector protein (gp10) N terminus were assembled into proheads in Escherichia coli and the particles tested for pRNA binding and DNA-gp3 packaging in vitro. The basic amino acid residues RKR at positions 3-5 of the gp10 N terminus were central to pRNA binding during assembly of an active DNA packaging motor. Conjugation of iron(S)-1-(p-bromoacetamidobenzyl) ethylenediaminetetraacetate (Fe-BABE) to residue S170C in the narrow end of the connector, near the N terminus, permitted hydroxyl radical probing of bound [32P]pRNA and identified two discrete sites proximal to this residue: the C-helix at the junction of the A, C and D helices, and the E helix and the CE loop/D loop of the intermolecular base pairing site.

KW - DNA packaging

KW - RNA binding

KW - bacteriophage ø29

KW - directed hydroxyl radical probing

KW - packaging motor assembly

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Alanine Scanning and Fe-BABE Probing of the Bacteriophage ø29 Prohead RNA-Connector Interaction

Abstract

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Keywords

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