• Dixon, Nicholas E.
• Oakley, Aaron J.
• Bancia, Bogdan
• Loscha, Karin
• Wilce, Matthew C. J.
• Schaeffer, Patrick M.
• Prosselkov, Pavel

Abstract

<p>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 λ-promoters, and the protein was purified in yields of 4-6mg/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 0.5 mM sample in 10 mM phosphate, pH 6.05, 0.1 M NaCl, recorded at 36°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 I4₁22, with unit cell parameters a = b = 142.2 Å, c = 192.1 Å, and diffracted beyond 2.7 Å resolution with synchrotron radiation.</p>

Topics

• impedance spectroscopy
• zinc
• Nuclear Magnetic Resonance spectroscopy
• crystallization
• space group