Alanine scanning and Fe-BABE probing of the bacteriophage ø29 prohead RNA-connector interaction.
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.
Atz
Rockney
R
Department of Diagnostic and Biological Sciences, University of Minnesota, Minneapolis, MN 55455, USA.
Ma
Shuhua
S
Gao
Jiali
J
Anderson
Dwight L
DL
Grimes
Shelley
S
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
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