Essential amino acid residues in the single-stranded DNA-binding protein of bacteriophage T7. Identification of the dimer interface.
Gene 2.5 of bacteriophage T7 is an essential gene that encodes a single-stranded DNA-binding protein. T7 phage with gene 2.5 deleted can grow only on Escherichia coli cells that express gene 2.5 from a plasmid. This complementation assay was used to screen for lethal mutations in gene 2.5. By screening a library of randomly mutated plasmids encoding gene 2.5, we identified 20 different single amino acid alterations in gene 2.5 protein that are lethal in vivo. The location of these essential residues within the three-dimensional structure of gene 2.5 protein assists in the identification of motifs in the protein. In this study we show that a subset of these alterations defines the dimer interface of gene 2.5 protein predicted by the crystal structure. Recombinantly expressed and purified gene 2.5 protein-P22L, gene 2.5 protein-F31S, and gene 2.5 protein-G36S do not form dimers at salt concentrations where the wild-type gene 2.5 protein exists as a dimer. The basis of the lethality of these mutations in vivo is not known because altered proteins retain the ability to bind single-stranded DNA, anneal complementary strands of DNA, and interact with T7 DNA polymerase.
Rezende
Lisa F
LF
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Hollis
Thomas
T
Ellenberger
Tom
T
Richardson
Charles C
CC
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017123:Bacteriophage T7 / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D020043:Genes, Essential; D005814:Genes, Viral; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000235:genetics
D017931:DNA Primers; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C085913:gene 2.5 protein, Enterobacteria phage T7