The role of tyrosine residues in the DNA-binding site of the Pf1 gene 5 protein.
The 144 amino acid gene 5 protein of bacteriophage Pf1 binds tightly and cooperatively to single-stranded DNA during replication of the phage genome. It has been suggested that aromatic amino acid side chains are important for this interaction, probably through base stacking with the DNA. We have analysed the accessibility of tyrosine residues in the DNA-protein complex, and their importance to the DNA-binding activity of the protein, by chemical modification and protection experiments using tetranitromethane. Tyrosines 21, 30 and 55 are surface accessible in the free protein but are protected from modification in the complex with phage DNA. Moreover, modification of these residues in the free protein abolishes the ability to bind to DNA or oligonucleotides, as judged by fluorescence spectroscopy and gel retardation analysis. Modification of the protein also results in the formation of an intersubunit covalent cross-link between Tyr55 and Phe76, suggesting that Phe76 is located within the DNA-binding cleft of the protein. It is proposed that residues 17-34 of the Pf1 gene 5 protein form a beta-hairpin analogous to the 'DNA-binding wing' of the fd and Ike gene 5 proteins. We suggest the existence of a single-stranded DNA binding motif, in which Tyr30 of the Pf1 protein is equivalent to the functionally important Tyr26 of the fd gene 5 protein.
Plyte
S E
SE
Biophysics Laboratories, Portsmouth Polytechnic, UK.
Kneale
G G
GG
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000502:physiology; D004279:DNA, Viral / Q000737:chemistry* / Q000502:physiology; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D011071:Poly T / Q000235:genetics; D011487:Protein Conformation; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D013774:Tetranitromethane; D014443:Tyrosine / Q000235:genetics / Q000502:physiology*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C067863:gene 5 protein, Pseudomonas phage Pf1; D011071:Poly T; D014443:Tyrosine; D013774:Tetranitromethane