Characterization of an RNA-binding domain in the bacteriophage phi 29 connector.
The connector of bacteriophage phi 29 is known to promote the viral prohead assembly, to bind DNA, and to drive DNA packaging into preformed viral shells in an RNA-dependent process. In this report, the phi 29 connector protein, p10, is shown to bind RNA in a sequence-independent fashion, and to possess an RNA recognition motif comprised approximately the region between residues 21 and 94 of the p10 sequence. Substitution mutants in specific amino acids of the RNA-binding domain obtained by site-directed mutagenesis showed that amino acids Phe23, His57, Phe59, and Tyr61 are critical for RNA binding and, subsequently, for DNA packaging into proheads. Proteolytic modified forms of the phi 29 connector have allowed us to conclude that the DNA- and RNA-binding domains are separated within the p10 sequence. It is also shown that RNA is stably associated to DNA-filled proheads during the DNA-packaging process.
Donate
L E
LE
Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientificas, Universidad Autónoma de Madrid, Spain.
Valpuesta
J M
JM
Mier
C
C
Rojo
F
F
Carrascosa
J L
JL
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D017103:Bacillus Phages / Q000235:genetics / Q000378:metabolism*; D001412:Bacillus subtilis / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D004279:DNA, Viral / Q000302:isolation & purification / Q000378:metabolism; D005814:Genes, Viral; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D012329:RNA, Bacterial / Q000302:isolation & purification / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000302:isolation & purification / Q000378:metabolism*
D004279:DNA, Viral; D012329:RNA, Bacterial; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D000255:Adenosine Triphosphate