Nucleotide and DNA-induced conformational changes in the bacteriophage T7 gene 4 protein.
The bacteriophage T7 gene 4 protein is a multifunctional enzyme that has DNA helicase, primase, and deoxyribonucleotide 5'-triphosphatase activities. Prior studies have shown that in the presence of dTTP or dTDP the gene 4 protein assembles into a functionally active hexamer prior to binding to single-stranded DNA. In this study, we have examined the effects of different nucleotide cofactors on the conformation of the gene 4 protein in the presence and absence of DNA. Gel retardation analysis, partial protease digestion, and DNA footprinting all suggest that the gene 4 protein undergoes a conformational change when dTTP is hydrolyzed to dTTP and that in the presence of dTDP the complex with DNA is more open or extended. We have also found that the dissociation constant of the gene 4 protein.DNA complex in the presence of dTDP was 10-fold lower than that determined in the presence of dTTP, further suggesting that these cofactors exerts different allosteric effects on the DNA-binding site of the gene 4 protein.
Yong
Y
Y
Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA.
Romano
L J
LJ
D017123:Bacteriophage T7 / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D018983:DNA Footprinting; D019915:DNA Primase; D004279:DNA, Viral / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli; D007700:Kinetics; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011487:Protein Conformation; D012316:RNA Nucleotidyltransferases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship; D013942:Thymine Nucleotides / Q000378:metabolism; D014357:Trypsin
D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013942:Thymine Nucleotides; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D014357:Trypsin; C024157:thymidine 5'-triphosphate