title: Expression, purification, crystallization, and NMR studies of the helicase interaction domain of Escherichia coli DnaG primase.
abstract: In Escherichia coli, the DnaG primase is the RNA polymerase that synthesizes RNA primers at replication forks. It is composed of three domains, a small N-terminal zinc-binding domain, a larger central domain responsible for RNA synthesis, and a C-terminal domain comprising residues 434-581 [DnaG(434-581)] that interact with the hexameric DnaB helicase. Presumably because of this interaction, it had not been possible previously to express the C-terminal domain in a stably transformed E. coli strain. This problem was overcome by expression of DnaG(434-581) under control of tandem bacteriophage lambda-promoters, and the protein was purified in yields of 4-6 mg/L of culture and studied by NMR. A TOCSY spectrum of a 2mM solution of the protein at pH 7.0, indicated that its structured core comprises residues 444-579. This was consistent with sequence conservation among most-closely related primases. Linewidths in a NOESY spectrum of a 0.5mM sample in 10mM phosphate, pH 6.05, 0.1M NaCl, recorded at 36 degrees C, indicated the protein to be monomeric. Crystals of selenomethionine-substituted DnaG(434-581) obtained by the hanging-drop vapor-diffusion method were body-centered tetragonal, space group I4(1)22, with unit cell parameters a=b=142.2A, c=192.1A, and diffracted beyond 2.7A resolution with synchrotron radiation.
authors.affiliation: Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia.
authors.forename: Karin
authors.initials: K
authors.lastname: Loscha
authors.forename: Aaron J
authors.initials: AJ
authors.lastname: Oakley
authors.forename: Bogdan
authors.initials: B
authors.lastname: Bancia
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Schaeffer
authors.forename: Pavel
authors.initials: P
authors.lastname: Prosselkov
authors.forename: Gottfried
authors.initials: G
authors.lastname: Otting
authors.forename: Matthew C J
authors.initials: MC
authors.lastname: Wilce
authors.forename: Nicholas E
authors.initials: NE
authors.lastname: Dixon
mesh_terms: D000595:Amino Acid Sequence; D003460:Crystallization; D018360:Crystallography, X-Ray; D003470:Culture Media; D019915:DNA Primase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
chemical_list: D003470:Culture Media; D011994:Recombinant Proteins; D019915:DNA Primase