title: Alanine scanning and Fe-BABE probing of the bacteriophage ø29 prohead RNA-connector interaction.
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 [(32)P]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.
authors: Department of Diagnostic and Biological Sciences, University of Minnesota, Minneapolis, MN 55455, USA.
authors: Rockney
authors: R
authors: Atz
authors: Shuhua
authors: S
authors: Ma
authors: Jiali
authors: J
authors: Gao
authors: Dwight L
authors: DL
authors: Anderson
authors: Shelley
authors: S
authors: Grimes
mesh_terms: D000409:Alanine / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001435:Bacteriophages / Q000378:metabolism*; D001483:Base Sequence; D036022:Capsid Proteins / Q000737:chemistry / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D042003:DNA Packaging; D004492:Edetic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D017665:Hydroxyl Radical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D036022:Capsid Proteins; D050505:Mutant Proteins; D012367:RNA, Viral; C097975:portal protein, bacteriophage phi29; D017665:Hydroxyl Radical; C042007:1-(4-bromoacetamidobenzyl)EDTA; D004492:Edetic Acid; D003545:Cysteine; D000409:Alanine