title: The primase active site is on the outside of the hexameric bacteriophage T7 gene 4 helicase-primase ring.
abstract: Gene 4 of bacteriophage T7 encodes a protein (gp4) that can translocate along single-stranded DNA, couple the unwinding of duplex DNA with the hydrolysis of dTTP, and catalyze the synthesis of short RNA oligoribonucleotides for use as primers by T7 DNA polymerase. Electron microscopic studies have shown that gp4 forms hexameric rings, and X-ray crystal structures of the gp4 helicase domain and of the highly homologous RNA polymerase domain of Escherichia coli DnaG have been determined. Earlier biochemical studies have shown that when single-stranded DNA is bound to the hexameric ring, the primase domain remains accessible to free DNA. Given these results, a model was suggested in which the primase active site in the gp4 hexamer is located on the outside of the hexameric ring. We have used electron microscopy and single-particle image analysis to examine T7 gp4, and have determined that the primase active site is located on the outside of the hexameric ring, and therefore provide direct structural support for this model.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia Health Sciences, Charlottesville, VA 22908-0733, USA.
authors.forename: M S
authors.initials: MS
authors.lastname: VanLoock
authors.forename: Y J
authors.initials: YJ
authors.lastname: Chen
authors.forename: X
authors.initials: X
authors.lastname: Yu
authors.forename: S S
authors.initials: SS
authors.lastname: Patel
authors.forename: E H
authors.initials: EH
authors.lastname: Egelman
mesh_terms: D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000201:enzymology*; D001665:Binding Sites; D019915:DNA Primase / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D008854:Microscopy, Electron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
chemical_list: D004277:DNA, Single-Stranded; D019915:DNA Primase