Acidic carboxyl-terminal domain of gene 2.5 protein of bacteriophage T7 is essential for protein-protein interactions.
The product of gene 2.5 protein of bacteriophage T7, a single-stranded DNA-binding protein, physically interacts with phage encoded DNA polymerase and primase/helicase proteins. A truncated gene 2.5 protein (GP2.5-delta 21C) was constructed by in vitro mutagenesis and lacks the 21 carboxyl-terminal amino acids found in wild-type gene 2.5 protein, 15 of which are acidic. GP2.5-delta 21C cannot substitute for wild-type gene 2.5 protein in vivo; the phage are not viable and exhibit less than 1% of the DNA synthesis observed in wild-type phage-infected cells. GP2.5-delta 21C has been purified to apparent homogeneity from cells overexpressing its cloned gene and has a conformation that differs from that of the wild-type gene 2.5 protein as judged by its circular dichroism spectra. Purified GP2.5-delta 21C retains its ability to bind to single-stranded DNA; the association constant of the protein for single-stranded DNA, determined by nitrocellulose filter binding, is 3.2 x 10(6) M-1 and is identical to that determined for wild-type gene 2.5 protein. However, GP2.5-delta 21C is a monomer in solution, whereas the wild-type protein exists as a dimer. GP2.5-delta 21C does not physically interact with T7 DNA polymerase as measured by affinity chromatography and fluorescent emission anisotropy. The mutant protein cannot stimulate T7 DNA polymerase activity on primed single-stranded DNA templates.
Kim
Y T
YT
Department of Biological Chemistry and Molecular Pharmacology, Harvard University Medical School, Boston, Massachusetts 02115.
Richardson
C C
CC
D017123:Bacteriophage T7 / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D017931:DNA Primers; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005814:Genes, Viral; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*
D017931:DNA Primers; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C085913:gene 2.5 protein, Enterobacteria phage T7; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase