PMID:14711519
| Citation |
Loscha, K, Oakley, AJ, Bancia, B, Schaeffer, PM, Prosselkov, P, Otting, G, Wilce, MC and Dixon, NE (2004) Expression, purification, crystallization, and NMR studies of the helicase interaction domain of Escherichia coli DnaG primase. Protein Expr. Purif. 33:304-10 |
|---|---|
| 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. |
| Links | |
| Keywords |
Amino Acid Sequence; Crystallization; Crystallography, X-Ray; Culture Media; DNA Primase/chemistry; DNA Primase/isolation & purification; DNA Primase/metabolism; Escherichia coli/enzymology; Escherichia coli/genetics; Gene Expression; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Plasmids; Recombinant Proteins/chemistry; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Restriction Mapping |
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