TY - JOUR
T1 - Expression, purification, crystallization, and NMR studies of the helicase interaction domain of Escherichia coli DnaG primase
AU - Loscha, Karin
AU - Oakley, Aaron J.
AU - Bancia, Bogdan
AU - Schaeffer, Patrick M.
AU - Prosselkov, Pavel
AU - Otting, Gottfried
AU - Wilce, Matthew C.J.
AU - Dixon, Nicholas E.
PY - 2004/1/1
Y1 - 2004/1/1
N2 - 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.
AB - 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.
KW - DNA replication
KW - DnaB helicase
KW - DnaG primase
KW - Primase-helicase interaction
KW - Protein structure
UR - http://www.scopus.com/inward/record.url?scp=0842331838&partnerID=8YFLogxK
U2 - 10.1016/j.pep.2003.10.001
DO - 10.1016/j.pep.2003.10.001
M3 - Article
C2 - 14711519
AN - SCOPUS:0842331838
SN - 1046-5928
VL - 33
SP - 304
EP - 310
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 2
ER -