Expression, purification, crystallization, and NMR studies of the helicase interaction domain of Escherichia coli DnaG primase
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Loscha, Karin; Oakley, Aaron; Bancia, Bogdan; Schaeffer, Patrick; Prosselkov, Pavel; Wilce, Matthew; Dixon, Nicholas; Otting, Gottfried
Description
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...[Show more]
dc.contributor.author | Loscha, Karin | |
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dc.contributor.author | Oakley, Aaron | |
dc.contributor.author | Bancia, Bogdan | |
dc.contributor.author | Schaeffer, Patrick | |
dc.contributor.author | Prosselkov, Pavel | |
dc.contributor.author | Wilce, Matthew | |
dc.contributor.author | Dixon, Nicholas | |
dc.contributor.author | Otting, Gottfried | |
dc.date.accessioned | 2015-12-07T22:52:26Z | |
dc.identifier.issn | 1046-5928 | |
dc.identifier.uri | http://hdl.handle.net/1885/27435 | |
dc.description.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 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. | |
dc.publisher | Academic Press | |
dc.source | Protein Expression and Purification | |
dc.subject | Keywords: DNA primase; recombinant protein; amino acid sequence; article; chemistry; crystallization; culture medium; enzymology; Escherichia coli; gene expression; genetics; isolation and purification; metabolism; molecular genetics; nuclear magnetic resonance spe DNA replication; DnaB helicase; DnaG primase; Primase-helicase interaction; Protein structure | |
dc.title | Expression, purification, crystallization, and NMR studies of the helicase interaction domain of Escherichia coli DnaG primase | |
dc.type | Journal article | |
local.description.notes | Imported from ARIES | |
local.identifier.citationvolume | 33 | |
dc.date.issued | 2004 | |
local.identifier.absfor | 030499 - Medicinal and Biomolecular Chemistry not elsewhere classified | |
local.identifier.ariespublication | u4217927xPUB51 | |
local.type.status | Published Version | |
local.contributor.affiliation | Loscha, Karin, College of Physical and Mathematical Sciences, ANU | |
local.contributor.affiliation | Oakley, Aaron, College of Physical and Mathematical Sciences, ANU | |
local.contributor.affiliation | Bancia, Bogdan, College of Physical and Mathematical Sciences, ANU | |
local.contributor.affiliation | Schaeffer, Patrick, College of Physical and Mathematical Sciences, ANU | |
local.contributor.affiliation | Prosselkov, Pavel, College of Physical and Mathematical Sciences, ANU | |
local.contributor.affiliation | Otting, Gottfried, College of Physical and Mathematical Sciences, ANU | |
local.contributor.affiliation | Wilce, Matthew, University of Western Australia | |
local.contributor.affiliation | Dixon, Nicholas, College of Physical and Mathematical Sciences, ANU | |
local.description.embargo | 2037-12-31 | |
local.bibliographicCitation.issue | 2 | |
local.bibliographicCitation.startpage | 304 | |
local.bibliographicCitation.lastpage | 310 | |
local.identifier.doi | 10.1016/j.pep.2003.10.001 | |
dc.date.updated | 2015-12-07T12:29:26Z | |
local.identifier.scopusID | 2-s2.0-0842331838 | |
Collections | ANU Research Publications |
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