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Expression, purification, crystallization, and NMR studies of the helicase interaction domain of Escherichia coli DnaG primase

Domenii publicaţii > Biologie + Tipuri publicaţii > Articol în revistã ştiinţificã

Autori: Loscha K1, Oakley AJ, Bancia B, Schaeffer PM, Prosselkov P, Otting G, Wilce MC, Dixon NE

Editorial: Protein Expr Purif. , 33, p.304-10, 2004.


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.

Cuvinte cheie: DNA replication, DnaG primase, DnaB helicase, Primase-helicase interaction, protein structure