Expression, purification, crystallization, and NMR studies of the helicase interaction domain of Escherichia coli DnaG primase.
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.
Loscha
Karin
K
Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia.
Oakley
Aaron J
AJ
Bancia
Bogdan
B
Schaeffer
Patrick M
PM
Prosselkov
Pavel
P
Otting
Gottfried
G
Wilce
Matthew C J
MC
Dixon
Nicholas E
NE
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
D003470:Culture Media; D011994:Recombinant Proteins; D019915:DNA Primase